Inhibition of Pathogens by Probiotic Bacteria

ABSTRACT

Compositions containing a lactic acid-producing bacterial strain, e.g.,  Bacillus coagulans  for inhibition of pathogenic bacterial infections. Spores or extracellular products produced by the bacterial strains are also useful as inhibitory agents.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 12/756,658 filedApr. 8, 2010, which is a divisional application of U.S. Ser. No.11/706,642, filed Feb. 13, 2007, which is a continuation of U.S. Ser.No. 11/005,897, filed on Dec. 6, 2004, which is a continuation of U.S.Ser. No. 09/708,870, filed on Nov. 8, 2000, which claims priority toU.S. Provisional Application Ser. No. 60/163,959, filed Nov. 8, 1999,entitled: “ISOLATION AND CHARACTERIZATION OF PROBIOTIC STRAINS OFBACILLUS COAGULANS”; and U.S. Provisional Application Ser. No.60/198,404, filed Apr. 19, 2000, entitled: “ADDITIONAL ISOLATION ANDCHARACTERIZATION OF PROBIOTIC STRAINS OF BACILLUS COAGULANS”; thedisclosures of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to methods of treatment and compositionsusing novel stains of probiotic organisms and/or their extracellularproducts in therapeutic compositions. More specifically, the presentinvention relates to the utilization of one or more species or strainsof probiotic bacteria and/or their extracellular products for thecontrol of gastrointestinal pathogens, including antibiotic-resistantspecies.

BACKGROUND OF THE INVENTION

The gastrointestinal microflora has been shown to play a number of vitalroles in maintaining gastrointestinal tract function and overallphysiological health. For example, the growth and metabolism of the manyindividual bacterial species inhabiting the gastrointestinal tractdepend primarily upon the substrates available to them, most of whichare derived from the diet. See, e.g., Gibson et al., 1995.Gastroenterology 106: 975-982; Christi, et al., 1992. Gut 33: 1234-1238;Gorbach, 1990. Ann. Med. 22: 37-41; Reid et al, 1990. Clin. Microbiol.Rev. 3: 335-344. These finding have led to attempts to modify thestructure and metabolic activities of the community through diet,primarily with probiotics, which are live microbial food supplements.The best-known probiotics are the lactic acid-producing bacteria (i.e.,Lactobacilli and Bifidobacteria), which are widely utilized in yogurtsand other dairy products. These probiotic organisms are non-pathogenicand non-toxigenic, retain viability during storage, and survive passagethrough the stomach and small intestine. Since probiotics do notpermanently colonize the host, they need to be ingested regularly forany health promoting properties to persist. Commercial probioticpreparations are generally comprised of mixtures of Lactobacilli andBifidobacteria, although yeast species such as Saccharomyces have alsobeen utilized.

There remains a need for the development of a highly efficacious,non-antibiotic-based therapeutic regimen which functions in acutetreatment scenarios, as well as prophylactically to mitigateantibiotic-resistant pathogens (e.g., antibiotic-resistant enterococci)in both humans and animals.

SUMMARY OF THE INVENTION

The invention provides compositions, therapeutic systems, and methods ofuse which exploit the discovery that novel lactic acid-producingbacterial strains (e.g., the novel strains of Bacillus coagulansdisclosed herein), or extracellular products thereof, possess theability to exhibit inhibitory activity in mitigating and preventing thegrowth and/or colonization rates of pathogenic bacterial, particularlygastrointestinal pathogens such as antibiotic-resistant pathogenicbacterial species including, but not limited to, Enterococccus,Clostridium, Escherichia, Klebsiella, Campylobacter, Peptococcus,Heliobacter, Hemophylus, Staphylococcus, Yersinia, Vibrio, Shigella,Salmonella, Streptococcus, Proteus, Pseudomonas, Toxoplasmosis, andRotovirus species, as well as mitigating the deleterious physiologicaleffects of the infection by the pathogen(s). Preferably, the bacteriaare probiotic. As currently defined, probiotic microorganisms are those,which confer a benefit when grow in a particular microenvironment by,e.g., directly inhibiting or preventing the growth of other biologicalorganisms within the same microenvironment. Examples of probioticorganisms include, but are not limited to, bacteria, which possess theability to grow within the gastrointestinal tract, at least temporarily,to displace or destroy pathogenic organisms, as well as providing otherbenefits to the host. See, e.g., Salminen et al, 1996. Antonie VanLeeuwenhoek 70: 347-358; Elmer et al, 1996. JAMA 275: 870-876; Rafter,1995. Scand J. Gastroenterol. 30: 497-502; Perdigon et al, 1995. J.Dairy Sci. 78: 1597-1606; Gandi, Townsend Lett. Doctors & Patients, pp.108-110, January 1994; Lidbeck et al, 1992. Eur. J. Cancer Prev. 1:341-353.

In addition, the novel strains of Bacillus coagulans disclosed hereinpossess biochemical and physiological characteristics which include, butare not limited to: (i) the production of the (L)+ optical isomer oflactic acid (propionic acid); (ii) have an optimal growth temperature ofbetween 20-44° C.; (iii) produces spores resistant to temperatures of upto approximately 90° C. which are able to germinate in a human or animalbody without specific inducement (e.g., heat-shock or otherenvironmental factors); (iv) the production of one or more extracellularproducts exhibiting probiotic activity which inhibits the growth ofbacteria, yeast, fungi, virus, or any combinations thereof; and/or (v)the ability to utilize a wide spectrum of substrates for proliferation.Preferably, the purified population of Bacillus coagulans has an optimalgrowth temperature of less than 45 degrees C. For example, the isolatedpopulation of Bacillus coagulans has an optimal growth temperature of 20degrees C., more preferably 30 degrees C., more preferably 35 degreesC., more preferably 36 degrees C., and most preferably 37 degrees C. Incontrast, previously identified populations of Bacillus coagulans havean optimal growth temperature of greater than 37 degrees C., e.g., anoptimal growth temperature of 45 degrees C. The strain grows at low pHsuch as pH conditions found in the gastrointestinal tract of a mammal,e.g., pH 2-5.

By purified or isolated preparation of a bacterial strain is meant thatthe preparation does not contain another bacterial species or strain ina quantity sufficient to interfere with the replication of thepreparation at a particular temperature. A purified or isolatedpreparation of a bacterial strain is made using standard methods, e.g.,plating at limiting dilution and temperature selection.

In one embodiment of the present invention, a therapeutic compositioncomprising Bacillus coagulans in a pharmaceutically-acceptable carriersuitable for oral administration to the gastrointestinal tract of ahuman or animal, is disclosed. In another embodiment, a Bacilluscoagulans strain is included in the therapeutic composition in the formof spores. In another embodiment, a Bacillus coagulans strain isincluded in the composition in the form of a dried or lyophilized cellmass.

An embodiment of the present invention involves the administration offrom approximately 1×10³ to 1×10¹⁴ CFU of viable, Bacillus coagulansvegetative bacteria or spore per day, more preferably from approximately1×10⁵ to 1×10¹¹, and preferably from approximately 5×10⁸ to 1×10¹⁰ CFUof viable, vegetative bacteria or spores per day. Where the condition tobe treated involves antibiotic-resistant digestive pathogens and thepatient is an adult, the typical dosage is approximately 1×10² to 1×10¹⁴CFU of viable, vegetative bacteria or spores per day, preferably fromapproximately 1×10⁸ to 1×10¹⁰, and more preferably from approximately2.5×10⁸ to 1×10¹⁰ CFU of viable, vegetative bacteria or spores per day.

In another aspect of the present invention, a composition comprising anextracellular product of Bacillus coagulans in apharmaceutically-acceptable carrier suitable for oral administration toa human or animal, is disclosed. In one embodiment, the extracellularproduct is a supernatant or filtrate of a culture of an isolatedBacillus coagulans strain. In another embodiment, the extracellularproduct is a semi-purified or purified, lyophilized supernatant orfiltrate of a culture of an isolated Bacillus coagulans strain. In apreferred embodiment, the extracellular product is the active agent(s)possessing the anti-microbial activity, which are isolated and purifiedfrom a supernatant or filtrate of a culture of an isolated Bacilluscoagulans strain.

The extracellular product is administered to a subject in a compositioncomprising a total concentration ratio of Bacillus coagulansextracellular product ranging from approximately 1% to 90% extracellularproduct with the remainder comprising the carrier or delivery component.The subject is preferably a mammal, e.g., a human. The bacteria and/orproducts derived from the bacteria are also suitable for veterinary use,e.g., to treat animals such as dogs and cats. A preferred embodimentcomprises a composition a total concentration ratio of Bacilluscoagulans extracellular product ranging from approximately 10% to 75%extracellular product with the remainder comprising the carrier ordelivery component.

The present invention is not limited solely to oral administration ofthe therapeutic compounds disclosed herein. Skin and or mucous membranesare treated using compositions containing Bacillus coagulans vegetativecells, spores, or extracellular products produced by vegetative cells.For example, the administration of the Bacillus coagulans strains,and/or the extracellular products thereof, aid in the mitigation ofvaginal pathogens, as well as reducing the incidence of relapse byre-population of the vagina with these probiotic, lactic acid-producingbacteria. The compositions are used to treat a condition characterizedby a reduction or absence of lactic acid-producing bacteria within thevagina, which condition is the a common etiology of both vaginal yeastinfections and bacterial vaginosis. Moreover, the use of such probioticbacterial strains are effective in the mitigation or prevention ofpathogens which are resistant to one or more antibiotics. Skin creams,lotions, gels, and the like, which contain Bacillus coagulans disclosedherein, and/or the extracellular products thereof, are effective in themitigation or prevention of pathogenic organisms on the skin, mucusmembrane, and cuticular tissues and further reduce the emergence ofantibiotic resistant pathogens. In addition to topical and oraladministration, the compositions are administered vaginally,intra-ocularly, intra-nasally, intra-otically, or buccally.

A further embodiment of the present invention involves the utilizationof probiotic organisms in livestock production, in which antibioticssuch as Vancomycin and Gentamicin are commonly used to stimulate healthand weight gain. Most, if not all, probiotic organisms are sensitive tothese two antibiotics and this fact has limited the potential use ofsuch microorganisms in the livestock industry. In addition, there aremany environmentally-related problems associated with the use ofantibiotics in livestock production. For example, antibiotic ladenanimal waste degrades very slowly and the antibiotic residue canpersist, further slowing biodegradation. With the addition of species ofbacteria that are resistant to Vancomycin, Gentamicin, and otherantibiotics, biodegradation is enhanced.

The present invention describes compositions, therapeutic systems, andmethods of use for inhibiting pathogen and/or parasite growth in thegastrointestinal tract and feces of animals. According to the invention,there is provided a composition comprising Bacillus coagulans vegetativecells or spores in a pharmaceutically- or nutritionally-acceptablecarrier suitable for oral administration to the digestive tract of ananimal. In another embodiment, the extracellular product from a Bacilluscoagulans culture is utilized, with or without Bacillus coagulansvegetative cells or spores.

In one embodiment, the bacteria is present in the composition at aconcentration of approximately 1×10³ to 1×10¹⁴ colony forming units(CFU)/gram, preferably approximately 1×10⁵ to 1×10¹² CFU/gram, whereasin other preferred embodiments the concentrations are approximately1×10⁹ to 1×10¹³ CFU/gram, approximately 1×10⁵ to 1×10⁷ CFU/g, orapproximately 1×10⁸ to 1×10⁹ CFU/gram.

In one embodiment, the bacteria is in a pharmaceutically acceptablecarrier suitable for oral administration to an animal, preferably, as apowdered food supplement, a variety of pelletized formulations, or aliquid formulation.

The invention also describes a therapeutic system for inhibitingpathogen and/or parasite growth in the gastrointestinal tract and/orfeces of an animal comprising a container comprising a label and acomposition as described herein, wherein said label comprisesinstructions for use of the composition for inhibiting pathogen and/orparasite growth.

The advantages of such a non-antibiotic, probiotic bacteria-basedtherapeutic regimen include, but are not limited to: (1) theadministration of the composition will result in the reduction of thecolonization rate of enterococci in the gastrointestinal tract; (ii) nocontribution to the development of antibiotic resistance; (iii) thecomposition can be used prophylactically to reduce the reservoir ofenterococci in hospitals, which will concomitantly reduce the chances ofhigh-risk patients from acquiring VRE; (iv) the dosage of thecomposition can be varied according to patient age, condition, etc; and(v) the composition may be utilized in food animal to reduce thedevelopment of further antibiotic resistance.

Unless defined otherwise, all scientific and technical terms used hereinhave the same meaning as commonly understood by those skilled in therelevant art. Unless mentioned otherwise, the techniques employed orcontemplated herein are standard methodologies well known to one ofordinary skill in the art. It should be understood that both theforegoing general description and the following detailed description areexemplary and explanatory only and are not restrictive of the presentinvention as claimed.

DESCRIPTION OF THE FIGURES

FIG. 1 is a bar graph showing the minimal and optimal culturetemperatures for the Bacillus coagulans 1% isolate (GBI-1); ATCC-=99%isolate (ATCC #31284); the 5937-20° C. isolate (GBI-20); and the5937-30° C. isolate (GBI-30), in either Trypticase Soy Broth (TSA) orGlucose Yeast Extract (GYE) media.

FIG. 2 is a bar graph showing the End-Point Kinetics of the 1% Bacilluscoagulans strain (GBI-1).

FIG. 3 is a bar graph showing the End-Point Kinetics of the ATCC-=99%Bacillus coagulans strain (ATCC #31284).

FIG. 4 is a bar graph showing the End-Point Kinetics of the 5937-20° C.Bacillus coagulans strain (GBI-20) and the 5937-30° C. Bacilluscoagulans strain (GBI-30) with TSB and GYE media.

FIG. 5 is a bar graph showing the End-Point Kinetics of the 5937-20° C.Bacillus coagulans strain (GBI-20) and the 5937-30° C. Bacilluscoagulans strain (GBI-30) with NB and BUGMB media.

FIG. 6 is a diagram showing the results from Alignment with otherBacillus species, Neighbor Joining Tree, and Concise Alignment analysisfor the Bacillus coagulans ATCC-=99% isolate (ATCC #31284).

FIG. 7 is a diagram showing the results from Alignment with otherBacillus species, Neighbor Joining Tree, and Concise Alignment analysisfor the Bacillus coagulans 20° C. isolate (GBI-20).

FIG. 8 is a diagram showing the results from Alignment with otherBacillus species, Neighbor Joining Tree, and Concise Alignment analysisfor the Bacillus coagulans 30° C. isolate (GBI-30).

FIG. 9 is a bar graph showing the results of the Aminopeptidase profilefor the Bacillus coagulans ATCC-=99% isolate (ATCC#31284).

FIG. 10 is a bar graph showing the results of the Aminopeptidase profilefor the Bacillus coagulans ATCC-=1% isolate (GBI-1).

FIG. 11 is a bar graph showing the results of the Aminopeptidase profilefor the Bacillus coagulans ATCC-=30° C. isolate (GBI-30).

FIG. 12 is a bar graph showing the results of the Aminopeptidase profilefor the Bacillus coagulans ATCC-20° C. isolate (GBI-20).

DETAILED DESCRIPTION OF THE INVENTION

Lactic acid-producing bacterial species, e.g., Lactobacillus,Bifidiobacterium, and the majority of Bacillus species have generallybeen thought to be unsuitable for colonization of the gut due to theirinstability in the harsh (i.e., acidic) pH environment of the bile,particularly human bile. However, Bacillus coagulans, including thenovel strains disclosed herein, was found to survive and colonize thegastrointestinal tract such as a bile environment and grown in this lowpH range. In particular, the human bile environment is different fromthe bile environment of animal models, and heretofore there has not beenany accurate descriptions of Bacillus coagulans growth in humangastrointestinal tract models.

With the current, dramatic increases in the number of bacterial strains,which exhibit resistance to one or more antibiotics, the development ofa non-antibiotic-based therapeutic regimen is of paramount importance.Prior to the disclosure of the present invention, there remained a needfor the development of a highly efficacious biorational therapy whichfunctions therapeutically in acute treatment scenarios, as well asprophylactically and in vector control applications to mitigateantibiotic-resistant pathogens (e.g., antibiotic-resistant enterococci)in both humans and animals, by the colonization (or re-colonization) ofthe gastrointestinal tract with probiotic microorganisms, which servesto reduce or prevent both the colonization rate and the potentialphysiologically deleterious effects due to the colonization ofantibiotic-resistant digestive pathogens.

Lactic acid producing bacteria are gram positive and vary in morphologyfrom long, slender rods to short coccobacilli, which frequently form“chains”. Their metabolism is fermentative; with some species beingaerotolerant (i.e., may utilize oxygen through the enzyme flavoproteinoxidase) while others are strictly anaerobic. Spore-forming lacticacid-producing bacteria are facultative anaerobes, whereas the rest arestrictly anaerobic. The growth of these bacteria is optimum at pH5.5-5.8, and the organisms have complex nutritional requirements foramino acids, peptides, nucleotide bases, vitamins, minerals, fattyacids, and carbohydrates. The lactic acid bacteria have the property ofproducing lactic acid from fermentable sugars. The genera Lactobacillus,Leuconostoc, Pediococcus, and Streptococcus are important members ofthis group. The taxonomy of lactic acid-producing bacteria has beenbased on the gram reaction and the production of lactic acid fromvarious fermentable carbohydrates. These groups include:

Homofermentative: produce more than 85% lactic acid from glucose.

Heterofermentative: produce only 50% lactic acid and considerableamounts of ethanol, acetic acid and carbon dioxide. Well-known are thehetero-fermentative species, which produce DL-lactic acid, acetic acidand carbon dioxide. These species, which have been used therapeutically,include: Lactobacillus acidophilus, Lactobacillus plantarum,Lactobacillus casei, Lactobacillus brevis, Lactobacillus delbruekii, andLactobacillus lactis.

While probiotic preparations were initially systematically evaluated fortheir effect on health and longevity in the early-1900's (see e.g.,Metchinikoff, Prolongation of Life, Willaim Heinermann, London 1910),their utilization has been markedly limited since the advent ofantibiotics in the 1950's to treat pathological microbes. See, e.g.,Winberg, et al, 1993. Pediatr. Nephrol. 7: 509-514; Malin et al, Ann.Nutr. Metab. 40: 137-145; and U.S. Pat. No. 5,176,911. Unfortunately,the majority of these early studies on probiosis were observationalrather than mechanistic in nature, and thus the processes responsiblefor many probiotic phenomena were not quantitatively elucidated.

There has been an increasing interest in the relationship betweenintestinal microflora and their effects on the health of the human host.The ecosystem of the human gastrointestinal tract is colonized by morethan 500 species of bacteria and represents an extremely complexmicroenvironment. The composition of the intestinal microflora isconstantly changing, being influenced by such factors as: diet, stress,age, and treatment with antibiotics and other drugs.

In order to provide the beneficial effects of lactic acid-producingbacteria, many manufacturers have been marketing various probioticpreparations. The reported health effects of these preparations includeeffectiveness in the treatment of a variety of disorders including, butnot limited to, colitis, constipation, diarrhea, flatulence, gastricacidity, gastroenteritis, gingivitis, hypercholesterolemia, hepaticencephalopathy and tumorigenesis, and in re-colonization of theintestine with beneficial flora after treatment with antibiotics.However, these reports are highly controversial due to such factors asdifferences in the viability of the implanted flora within thegastrointestinal tract. Successful utilization depends upon thefollowing factors: (i) a high count of viable organisms retaining theirviability during manufacturing into dosage forms and subsequent storage;(ii) survival of these lactic acid producing bacteria, once ingested, inthe acidic gastric secretions and their passage into the intestine; and(iii) the production of a sufficient quantity of metabolitesantagonistic to pathogens (e.g., L(+) (dextrorotatory) lactic acid andbacteriocins).

Previously, numerous species of Lactobacilli have been examinedincluding, but not limited to, Lactobacillus bulgaricus, Lactobacillusbifidus, Lactobacillus acidophilus, Lactobacillus casei, andLactobacillus brevis. Interestingly, however, Lactobacillus acidophilus,long regarded as the best candidate for therapeutic use, has beensubsequently shown to be highly ineffective as a probiotic organism forthe re-colonization of the gastrointestinal tract and in the alleviationof gastrointestinal disorders. Moreover, this bacterial strain producesD(−) (levorotatory) lactic acid, which is not an effective antagonisticagent and may potentially introduce metabolic disturbances. In view ofthis fact, the World Health Organization (WHO) has recommendedrestricted intake of D(−) lactic acid for adults and total avoidance ininfant nutrition.

It is now known that probiotic bacteria mitigate or prevent the growthof putrefactive or pathogenic microorganisms by the process ofcompetitive inhibition, through the generation of a non-physiologicallyconducive acidic environment (i.e., through the production of lactic orother biological acids) and/or by the production of antibiotic-likesubstances (i.e., bacteriocins), which are responsible for thebacteria's anti-microbial effects. See, e.g., Klaenhammer, 1993. FEMSMicrobiol. Rev. 12: 39-85; Barefoot et al., 1993. J. Diary Sci. 76:2366-2379. For example, selected Lactobacillus strains, which produceantibiotics, have been demonstrated as effective for the treatment ofinfections, sinusitis, hemorrhoids, dental inflammations, and variousother inflammatory conditions. See, e.g., U.S. Pat. No. 5,439,995.Similarly, Lactobacillus reuteri has been shown to produce antibioticswhich possess anti-microbial activity against Gram negative and Grampositive bacteria, yeast, and various protozoan. See, e.g., U.S. Pat.Nos. 5,413,960 and 5,439,678. Additionally, the proteolytic, lipolytic,and β-galactosidase activities of probiotic bacteria have also beenshown to improve the digestibility and assimilation of ingestednutrients, thereby rendering them valuable in convalescent/geriatricnutrition and as adjuncts to antibiotic therapy.

Probiotics have also been shown to possess anti-mutagenic properties.For example, Gram positive and Gram negative bacteria have beendemonstrated to bind mutagenic pyrolysates which are produced duringcooking at a high temperature. Studies performed with lactic acidproducing bacteria has shown that these bacteria may be either living ordead, due to the fact that the process occurs by adsorption of mutagenicpyrolysates to the carbohydrate polymers present in the bacterial cellwall. See, e.g., Zang, et al., 1990. J. Dairy Sci. 73: 2702-2710.Lactobacilli have also been shown to degrade carcinogens (e.g.,N-nitrosamines), which may serve an important role if the process issubsequently found to occur at the level of the mucosal surface. See,e.g., Rowland and Grasso, 1986. Appl. Microbiol. 29: 7-12. Additionally,the co-administration of lactulose and Bifidobacteria longum to ratsinjected with the carcinogen azoxymethane was demonstrated to reduceintestinal aberrant crypt foci, which are generally considered to bepre-neoplastic markers. See, e.g., Challa, et al., 1997. Carcinogenesis18: 5175-21. Purified cell walls of Bifidobacteria may also possessanti-tumorigenic activities in that the cell wall of Bifidobacteriainfantis induces the activation of phagocytes to destroy growing tumorcells. See, e.g., Sekine, et al., 1994. Bifidobacteria and Microflora13: 65-77. Bifidobacteria probiotics have also been shown to reducecolon carcinogenesis induced by 1,2-dimethylhydrazine in mice whenconcomitantly administered with fructo-oligosaccharides (FOS; see e.g.,Koo and Rao, 1991. Nutrit. Rev. 51: 137-146), as well as inhibitingliver and mammary tumors in rats (see e.g., Reddy and Rivenson, 1993.Cancer Res. 53: 3914-3918). Interestingly, populations at high risk forcolon cancer have been found to harbor gut flora, which efficientlymetabolize steroids and hydrolyze glucuronides while concomitantlyproducing carcinogens (e.g., nitrosamines). A diet containing largeconcentrations of viable, lactic acid-producing bacteria was found tosignificantly lower these deleterious bacterial-mediated activities insuch individuals.

It has also been demonstrated that the microbiota of thegastrointestinal tract affects both mucosal and systemic immunity withinthe host. See, e.g., Famularo, et al., Stimulation of Immunity byProbiotics. In: Probiotics: Therapeutic and Other Beneficial Effects.pg. 133-161. (Fuller, R., ed. Chapman and Hall, 1997). The intestinalepithelial cells, blood leukocytes, B- and T-lymphocytes, and accessorycells of the immune system have all been implicated in theaforementioned immunity. See, e.g., Schiffrin, et al., 1997. Am. J.Clin. Nutr. 66(suppl): 5-20S. Other bacterial metabolic products, whichpossess immunomodulatory properties, include: endotoxiclipopolysaccharide, peptidoglycans, and lipoteichoic acids. See, e.g.,Standiford, 1994. Infect. Linmun. 62: 119-125. Accordingly, probioticorganisms are thought to interact with the immune system at many levelsincluding, but not limited to: cytokine production, mononuclear cellproliferation, macrophage phagocytosis and killing, modulation ofautoimmunity, immunity to bacterial and protozoan pathogens, and thelike. See, e.g., Matsumara, et al., 1992. Animal Sci. Technol. (Jpn) 63:1157-1159; Solis-Pereyra and Lemmonier, 1993. Nutr. Res. 13: 1127-1140.Lactobacillus strains have also been found to markedly effect changes ininflammatory and immunological responses including, but not limited to,a reduction in colonic inflammatory infiltration without eliciting asimilar reduction in the numbers of B- and T-lymphocytes. See, e.g., DeSimone, et al., 1992. Immunopharmacol. Immunotoxicol. 14: 331-340.

While the attachment of probiotics to the gastrointestinal epithelium isan important determinant of their ability to modify host immunereactivity, this is not a universal property of Lactobacilli orBifidobacteria, nor is it essential for successful probiosis. See, e.g.,Fuller, 1989. J. Appl. Bacteriol. 66: 365-378. For example, adherence ofLactobacillus acidophilus and some Bifidobacteria to humanenterocyte-like CACO-2 cells has been demonstrated to prevent binding ofenterotoxigenic and enteropathogenic Escherichia coli, as well asSalmonella typhimurium and Yersinia pseudotuberculosis. See, e.g.,Bernet, et al., 1994. Gut 35: 483-489; Bernet, et al., 1993. Appl.Environ. Microbiol. 59: 4121-4128.

While the gastrointestinal microflora presents a microbial-based barrierto invading organisms, pathogens often become established when theintegrity of the microbiota is impaired through stress, illness,antibiotic treatment, changes in diet, or physiological alterationswithin the G.I. tract. For example, Bifidobacteria are known to beinvolved in resisting the colonization of pathogens in the largeintestine. See, e.g., Yamazaki, et al., 1982. Bifidobacteria andMicroflora 1: 55-60. Similarly, the administration of Bifidobacteriabreve to children with gastroenteritis eradicated the causativepathogenic bacteria (i.e., Campylobacter jejuni) from their stools (seee.g., Tojo, 1987. Acta Pediatr. Jpn. 29: 160-167) and supplementation ofinfant formula milk with Bifidobacteria bifidum and Streptococcusthermophilus was found to reduce rotavirus shedding and episodes ofdiarrhea in children who were hospitalized (see e.g., Saavedra, 1994.The Lancet 344: 1046-109.

Additionally, lactic acid producing bacteria also are able to colonizethe skin and mucus membranes, and may be used either prophylactically ortherapeutically to control bacterial infections. For example, lacticacid producing bacteria are able to utilize glycogen in the vaginalepithelial cells to produce lactic acid, which keeps the pH of thisenvironment in the range 4.0 to 4.5. This acidic environment is notconducive for the growth of pathogens such as Candida albicans,Gardnerella vaginalis, and various non-specific bacteria, which areresponsible for vaginal infections. There is a large body ofquantitative evidence, which has demonstrated that the depletion ofthese lactic acid-producing bacteria is the cause and effectrelationship in fungal and bacterial gynecological diseases.

Antibiotic Administration and Production of MultipleAntibiotic-Resistant Pathogenic Bacterial Strains

Antibiotics are widely used to control pathogenic microorganisms in bothhumans and animals. Unfortunately, the widespread use of anti-microbialagents, especially broad-spectrum antibiotics, has resulted in a numberof serious clinical consequences. For example, antibiotics often killbeneficial, non-pathogenic microorganisms (i.e., flora) within thegastrointestinal tract, which contribute to digestive function andhealth. Accordingly, relapse (the return of infections and theirassociated symptoms) and secondary opportunistic infections often resultfrom the depletion of lactic acid producing and other beneficial florawithin the gastrointestinal tract.

Unfortunately, most, if not all, lactic acid-producing or probioticbacteria are extremely sensitive to common antibiotic compounds.Accordingly, during a normal course of antibiotic therapy, manyindividuals develop a number of deleterious physiological side-effectsincluding: diarrhea, intestinal cramping, and sometimes constipation.These side-effects are primarily due to the non-selective action ofantibiotics, as antibiotics do not possess the ability to discriminatebetween beneficial, non-pathogenic and pathogenic bacteria, bothbacterial types are killed by these agents. Thus, individuals takingantibiotics offer suffer from gastrointestinal problems as a result ofthe beneficial microorganisms (i.e., intestinal flora), which normallycolonize the gastrointestinal tract, being killed or severelyattenuated. The resulting change in the composition of the intestinalflora can result in vitamin deficiencies when the vitamin-producingintestinal bacteria are killed, diarrhea and dehydration and, moreseriously, illness should a pathogenic organism overgrow and replace theremaining beneficial gastrointestinal bacteria.

Another deleterious result of indiscriminate use of anti-microbialagents is the generation of multiple antibiotic-resistant pathogenicbacterial stains. See, e.g., Mitchell, 1998. The Lancet 352: 462-463.For example, a meticillin-resistant Staphylococcus aurous (MRSA) strainwas responsible for over 50 deaths in a single Australian hospital. See,Shannon, 1998. Lancet 352: 490-491. However, these initial reports ofMRSA infections have been over-shadowed by the more recent outbreaks ofmultiple drug resistant (MDR) strains of Enterococci, includingvancomycin-resistant Enterococci (VRE). Vancomycin is generally regardedas an antibiotic of “last resort”. The development of such resistancehas led to numerous reports of systemic infections which remaineduntreatable with conventional antibiotic therapies.

Multiple Drug-Resistant Enterococci

Vancomycin-resistant enterococci (VRE) have emerged as importantnosocomial pathogens in the past decade. First reported in the UnitedStates in 1989, these organisms have rapidly spread throughout thecountry. VRE, particularly Enterococcus faecium strains, are oftenresistant to all antibiotics that are effective for treatment ofsusceptible enterococci. This situation has left clinicians treating VREinfections with either sub-optimal bacteriostatic agents (e.g.,chloramphenicol) or no therapeutic options. Efforts to limit the spreadof VRE through infection control measures and reduction of vancomycinuse have had a limited effect.

Intestinal colonization with VRE is the most important source for spreadof these organisms. Most patients harboring VRE have a-symptomaticintestinal colonization that may persist for months. These patients areat risk to develop VRE infection and are a potential source for spreadto healthcare workers, the environment, and to other patients. Theinfection control measures that are implemented to minimize the spreadof VRE are expensive and inconvenient for patients, family members, andhealthcare workers.

Recent studies have demonstrated a profound potential for lactic acidproducing Bacillus coagulans species, especially the novel strains ofBacillus coagulans disclosed herein, for use in bio-rational therapiesfor the prophylactic or therapeutic treatment of antibiotic-resistantdigestive pathogens. With the present state of emerging infectiousdisease and antibiotic-resistance, new therapies and new ways ofthinking about controlling pathogens are required. Antibiotics, in someapplications, have outlived their usefulness when considering themassive reservoir of new and antibiotic resistant strains that haveresulted from the misuse of antibiotics in the healthcare setting and“growth factors” in production animal operations.

Enterococci, leading causes of nosocomial bacteremia, surgical woundinfection, and urinary tract infection, are becoming resistant to manyand sometimes all standard therapies. New rapid surveillance methods arehighlighting the importance of examining enterococcal isolates at thespecies level. Most enterococcal infections are caused by Enterococcusfaecalis, which are more likely to express traits related to overtvirulence but, at least for the moment, also more likely to retainsensitivity to at least one effective antibiotic. The remaininginfections are mostly caused by Enterococcus faecium, a speciesvirtually devoid of known overt pathogenic traits but more likely to beresistant to even antibiotics of last resort. Effective control ofmultiple drug-resistant Enterococci will require: (i) betterunderstanding of the interaction between Enterococci, the environment,and humans; (ii) far more prudent antibiotic use; (iii) better contactisolation in hospitals and other patient care environments; (iv)improved surveillance; and, most importantly, (v) the development of newtherapeutic paradigms (e.g., non-antibiotic-based) which are lessvulnerable to the cycle of drug introduction and drug resistance.

Two types of Enterococci cause infections: (i) those originating frompatients' native flora, which are unlikely to possess resistance beyondthat which is intrinsic to the genus and are unlikely to be spread, and(ii) isolates that possess multiple antibiotic resistance traits and arecapable of nosocomial transmission. The therapeutic challenge ofmultiple-drug resistant (MDR) Enterococci (i.e., those strains withsignificant resistance to two or more antibiotics, often including, butnot limited to, vancomycin), has brought their role as importantnosocomial pathogens into sharper focus.

During the last decade, enterococci have become recognized as leadingcauses of nosocomial bacteremia, surgical wound infection, and urinarytract infection. Two types of enterococci are generally found to beassociated with causing infections: (i) those originating from patients'native flora, which are unlikely to possess resistance beyond thatintrinsic to the genus and are unlikely to be spread from bed to bed;and (ii) isolates that possess multiple antibiotic resistance traits andare capable of nosocomial transmission. The therapeutic challenge ofmultiple-drug resistant (MDR) enterococci (i.e., those strains withsignificant resistance to two or more antibiotics, often including, butnot limited to, vancomycin) has brought their role as importantnosocomial pathogens into sharper focus.

Enterococci normally inhabit the bowel and may be found in the intestineof nearly all animals, from cockroaches to humans. In humans, typicalconcentrations of enterococci in stool are up to 1×10⁸ CFU per gram.See, e.g., Rice, et al., 1995. Occurrence of high-level aminoglycosideresistance in environmental isolates of enterococci. Appl. Environ.Microbiol. 61: 374-376. The predominant species inhabiting the intestinevaries. In Europe, the United States, and the Far East, Enterococcusfaecalis predominates in some instances, and Enterococcus faecium inothers. Moreover, of the 4 or more known enterococcal species (see,e.g., Devriese, et al., 1993. Phenotypic identification of the genusEnterococcus and differentiation of phylogenetically distinctenterococcal species and species groups. J. Appl. Bacteria 75: 399-408),only Enterococcus faecalis and Enterococcus faecium commonly colonizeand infect humans in detectable numbers with Enterococcus faecalis beingisolated from approximately 80% of human infections, and Enterococcusfaecium from the remaining individuals.

Enterococci are exceedingly hardy and tolerate a wide variety of growthconditions, including temperatures of 10° C. to 45° C., and hypotonic,hypertonic, acidic, or alkaline environments. Sodium azide andconcentrated bile salts, which inhibit or kill most microorganisms, aretolerated by Enterococci and are actually used as selective agents inagar-based media. As facultative organisms, enterococci grow underreduced or oxygenated conditions, although enterococci are usuallyconsidered strict fermenters because they lack a Kreb's Cycle andrespiratory chain. However, Enterococcus faecalis is an exception sinceexogenous hemin can be used to produce d, b, and o type cytochromes.Enterococcus faecalis cytochromes are only expressed under aerobicconditions in the presence of exogenous hemin and, therefore, maypromote the colonization of inappropriate sites.

Enterococci are also intrinsically resistant to many antibiotics. Unlikeacquired resistance and virulence traits, which are usually transposonor plasmid encoded, intrinsic resistance is based in chromosomal genes,which typically are non-transferable. Penicillin, ampicillin,piperacillin, imipenem, and vancomycin are among the few antibioticsthat show consistent inhibitory, but not bactericidal, activity againstEnterococcus faecalis. Enterococcus faecium is less susceptible toβ-lactam antibiotics than Enterococcus faecalis because thepenicillin-binding proteins of the former have markedly lower affinitiesfor the antibiotics. The first reports of strains highly resistant topenicillin began to initially appear in the 1980s. See, e.g., Bush, etal., 1989. High-level penicillin resistance among isolates ofenterococci: implications for treatment of enterococcal infections. Ann.Intern. Med. 110: 515-520; Sapico, et al., 1989. Enterococci highlyresistant to penicillin and ampicillin an emerging clinical problem. J.Clin. Microbiol. 27: 2091-2095.

As will be more-fully discussed below, Enterococci often acquireantibiotic-resistance through exchange of resistance-encoding genescarried on conjugative transposons, pheromone-responsive plasmids, andother broad-host-range plasmids. The past two decades have witnessed therapid emergence of MDR enterococci. High-level gentamicin resistance wasinitially reported in 1979 (see, e.g., Horodniceanu, et al., 1979.High-level, plasmid-borne resistance to gentamicin in Streptococcusfaecalis sub-sp. zymogens. Antimicrob. Agents Chemother. 16: 686-689.),and was quickly followed by numerous reports of nosocomial infection inthe 1980's (see, e.g., Zervos, et al., 1987. Nosocomial infection bygentamicin-resistant Streptococcus faecalis: an epidemiologic study.Ann. Intern. Med. 106: 687-691). Simultaneously, sporadic outbreaks ofnosocomial Enterococcus faecalis and Enterococcus facium infectionappeared with penicillin resistance due to β-lactamase production;however, such isolates remain relatively rare. Finally, MDR enterococcithat had lost susceptibility to vancomycin were reported in Europe andthe United States. See, e.g., Sahm, et al., 1989. In-vitrosusceptibility studies of vancomycin-resistant Enterococcus faecalis.Antimicrob. Agents Chemother. 33: 1588-1591.

Among several phenotypes for vancomycin-resistant enterococci, VanA(resistance to vancomycin and teicoplanin) and VanB (resistance tovancomycin alone) are most common. In the United States, VanA and VanBaccount for approximately 60% and 40% of vancomycin-resistantEnterococci (VRE) isolates, respectively. See, e.g., Clark, et al.,1993. Characterization of glycopeptide-resistant Enterococci from U.S.hospitals. Antimicrob. Agents Chemother. 37: 2311-2317. Inducible genesencoding these phenotypes alter cell wall synthesis and render strainsresistant to glycopeptides. It has been demonstrated, in the laboratory,that these genes can be transferred from Enterococci to other bacteria.See, e.g., Arthur, et al., 1993. Genetics and mechanisms forglycopeptide-resistance in Enterococci. Antimicrob. Agents Chemother.37: 1563-1571. For example, Staphylococcus aureus has been renderedvancomycin-resistant through apparent transfer of resistance fromEnterococcus faecalis.

As previously discussed, most enterococci have naturally occurring orinherent resistance to various drugs, including cephalosporins and thesemisynthetic penicihinase-resistant penicillins (e.g., oxacillin) andclinically-achievable concentrations of clindamycin and aminoglycosides.Compared with streptococci, most enterococci are relatively resistant topenicillin, ampicillin, and the pseudopenicillins. Many enterococci arealso tolerant to the killing effects of cell-wall active agents (e.g.,ampicillin, vancomycin, etc.); although recent data suggest that thisproperty may not be inherent, but rather acquired after exposure toantibiotics. For example, the inherent in vivo resistance ofEnterococcus faecalis to trimethoprim-sulfamethoxazole, may explain thelack of efficacy in animal models. Moreover, bactericidal activityagainst Enterococcus faecalis seems unreliable and very methoddependent. In animal models, this combination has not shown goodactivity and is not generally accepted as an effective anti-enterococcaltherapy, especially for systemic infections.

In addition to natural resistance to many agents, enterococci have alsodeveloped plasmid- and transposon-mediated resistance to thetetracyclines (e.g., minocycline and doxycycline); erythromycin (e.g.,azithromycin and clarithromycin); chloramphenicol; high levels oftrimethoprim; and high levels of clindamycin. The propensity ofEnterococcus faecalis to acquire multiple antibiotic-resistance traitsmay result from a variety of distinctly different mechanisms forconjugation.

The best-characterized system of conjugation involves pheromoneoligopeptides and pheromone-responsive plasmids. See, e.g., Clewell andKeith, 1989. Sex pheromones and plasmid transferin Enterococcusfaecalis. Plasmid 21: 175-184. In this conjugation system, strains ofEnterococcus faecalis typically secrete into the culture medium a numberof different small, oligopeptide sex pheromones which are specific fordifferent types of plasmids. When a cell containing apheromone-responsive plasmid (i.e., the potential donor cell) comes intocontact with its corresponding pheromone, transcription of a gene on theplasmid is turned on, resulting in the synthesis of an aggregationsubstance on the surface of its cell membrane. When the donor cell, inturn, comes in contact with another Enterococcus faecalis bacterium, theaggregation substance (which contains two Arg-Gly-Asp motifs adheres tothe binding substance on the surface of most Enterococcus faecaliscells, causing them to aggregate. By a process, not yetwell-characterized, the pheromone-responsive plasmid can then transferfrom the donor bacterium to the other (recipient) bacterium. Once therecipient cell has acquired this particular plasmid, the synthesis ofthe corresponding sex pheromone is shut-off to prevent self-aggregation.This system of conjugation, which occurs primarily in Enterococcusfaecalis, is a highly efficient means of plasmid transfer.

Another system of conjugation, also not well-characterized, involvesbroad host-range plasmids that can transfer among species of enterococciand other gram-positive organisms such as streptococci andstaphylococci. See, e.g., Clewell, 1981. Plasmids, drug resistance, andgene transfer in the genus Streptococcus. Microbiol. Rev. 45: 409-436.The transfer frequency is generally much lower than with the pheromonesystem. Since staphylococci, streptococci, and enterococci share anumber of resistance genes, these broad host-range plasmids may be amechanism by which some of these resistance genes have spread amongdifferent genera.

A third type of conjugation, which involves conjugative transposons, mayalso explain the spread of resistance genes to many different species.See, e.g., Clewell, 1986. Conjugative transposons and the disseminationof antibiotic resistance in streptococci. Annu. Rev. Microbial. 40:635-659. As opposed to ordinary transposons, which can jump within acell from one DNA location to another, conjugative transposons alsoencode the ability to bring about conjugation between differentbacterial cells. Since plasmids typically require rather complexmachinery for replication (often depending on successful interactionswith host proteins) and must face additional problems of surfaceexclusion and incompatibility, conjugative transposons (which do notreplicate, but instead insert into the chromosome or into a plasmid ofthe new host) appear to be an even more efficient and far-reaching wayof disseminating a resistance gene. This may explain why the tetM geneof the conjugative transposon Tn916 has spread beyond the gram-positivespecies into gram-negative organisms, including gonococci andmeningococci, as well as into mycoplasma and ureaplasma. See, e.g.,Roberts, 1990. Characterization of the TetM determinants in urogenitaland respiratory bacteria. Antimicrob. Agents Chemother. 34: 476-478.Other resistance genes, including those encoding resistance toerythromycin and kanamycin, are also found on conjugative transposons;these frequently contain or are related to Tn916. Such transposons mayhave evolved from a Tn916 ancestor; their emergence suggests thepossibility of further dissemination of resistance among gram-positiveorganisms. Particularly ominous are reports of the vanB gene clusterwithin large conjugative chromosomal elements that appear similar, atleast in function, to conjugative transposons.

Epidemiology of Multiple Drug-Resistant Enterococci

Colonization and infection with MDR enterococci occur worldwide. Earlyreports showed that in the United States, the percentage of nosocomialinfections caused by VRE increased more than 20-fold (i.e., from 0.3% to7.9%) between 1989 and 1993, indicating rapid dissemination. Newdatabase technologies, such as The Surveillance Network (TSN)Database-USA, now permit the assessment of resistance profiles accordingto species. TSN Database collects and compiles data daily from more than100 clinical laboratories within the United States, identifies potentiallaboratory testing errors, and detects emergence of resistance profilesand mechanisms that pose a public health threat (e.g.,vancomycin-resistant staphylococci). Data collected by the TSN Databasebetween 1995 and Sep. 1, 1997 were analyzed to determine whether theearlier increase in vancomycin resistance was unique to vancomycin,whether it represented a continuing trend, and whether speciation isquantifiably important in analyzing this trend. Enterococci faecalisresistance to ampicillin and vancomycin is uncommon. Little change inresistance prevalence occurred from 1995 to 1997. In contrast,Enterococcus faecium vancomycin and ampicillin resistance increasedalarmingly. For example, in 1997, 771 (52%) of 1,482 Enterococcusfaecium isolates exhibited vancomycin resistance, and 1,220 (83%) of1,474 isolates exhibited ampicillin resistance. Enterococci faeciumresistance notwithstanding, Enterococci faecalis remained by far themost commonly encountered of the two Enterococcal species in TSNDatabase. Enterococci faecalis to Enterococci faecium total isolateswere approximately 4:1; blood isolates 3:1; and urine isolates 5:1. Thisobservation underscores important differences in the survival strategiesand likelihood of therapeutic success, critical factors usually obscuredby lumping the organisms together as Enterococcus species orenterococci.

Widespread emergence and dissemination of ampicillin and vancomycinresistance in Enterococcus faecalis would significantly confound thecurrent therapeutic dilemma. There is little reason to suspect thatvancomycin and ampicillin resistances only provide selective advantagefor the species faecium and not faecalis. The relative absence of theseresistances in Enterococcus faecalis may simply reflect a momentary lackof penetrance and equilibration of the traits. Because of theseimportant differences between the two species, meaningful surveillanceof Enterococcal resistance must include species identification.

It has been demonstrated that enterococci account for approximately110,000 urinary tract infections, 25,000 cases of bacteremia, 40,000wound infections, and 1,100 cases of endocarditis annually in the UnitedStates, with most of these infections occurring in hospitals.Enterococcal infection-related deaths have been difficult to ascertain,due to the fact that severe co-morbid illnesses are common. However,enterococcal sepsis is implicated in up to 50% of fatal cases. Moreover,several recent case-control and historical cohort studies have shownthat death risk associated with antibiotic-resistant enterococcalbacteremia is markedly higher than with susceptible enterococcalbacteremia. This trend is predicted to increase, as MDR isolates becomemore prevalent.

Although exact modes of nosocomial transmission for MDR Enterococci aredifficult to ascertain, molecular microbiologic and epidemiologicalevidence strongly suggest spread between patients, probably on the handsof health-care providers or medical devices, and between hospitals bypatients with prolonged intestinal colonization. Numerous outbreaks ofMDR Enterococci have been reported; and all but two were due toEnterococcus faecium. This disparity, particularly in view of the highernumbers of clinical Enterococcus faecalis isolates, may reflect areporting bias due to the novelty of the combinations of resistance thatoccur in Enterococcus faecium. When isolates from outbreaks of MDREnterococci have been analyzed by DNA sequencing, more than half havebeen demonstrated to involve clonally-related isolates.

Prior treatment with antibiotics is common in nearly all patientscolonized or infected with MDR Enterococci. See, e.g., Montecalvo, etal., 1994. Outbreak of vancomycin-, ampicillin-, andaminoglycoside-resistant Enterococcus faecium bacteremia in an adultoncology unit. Antimicrob. Agents Chemother. 38: 1363-1367. Other riskfactors include prolonged hospitalization; high severity of illnessscore; intra-abdominal surgery; renal insufficiency; enteral tubefeedings; and exposure to specific hospital units, nurses, orcontaminated objects and surfaces within patient-care areas.

Antibiotics may promote colonization and infection with MDR Enterococciby at least two mechanisms. First, many broad spectrum antibiotics havelittle or no anti-enterococcal activity, and administration commonlyleads to overgrowth of susceptible (or resistant) Enterococci at sitesat risk for infection. Second, most antibiotics substantially reduce thenormal resistance of the intestinal tract to colonization by exogenousorganisms. Colonization resistance results primarily from the “limitingaction” of the normal anaerobic flora, and to a lesser extent from anintact mucosa, gastric acid secretion, intestinal motility, andintestinal-associated immunity. Antibiotic-induced alterations in theprotective flora of the intestine serve as a catalyst for colonizationwith exogenous pathogens such as MDR Enterococci. Antibiotic restrictionprograms would be more effective if they included prudent prescribing ofall antibiotics, not just single agents (e.g., vancomycin). For example,use of this approach substantially decreased intestinal colonizationwith VRE in one hospital pharmacy that restricted vancomycin,cefotaxime, and clindamycin use. See, e.g., Quale, et al., 1996.Manipulation of a hospital antimicrobial formulary t control an outbreakof vancomycin-resistant enterococci. clin. Infect. Dis. 23:1020-1025.

Vancomycin Resistance Genetic Elements

In recent years there has been an alarming emergence among Enterococciof acquired resistance to vancomycin. Vancomycin had been in clinicaluse since the 1950s, although it was not heavily used until thelate-1970s and particularly the 1980s. Because multiple bacterial genesare involved in the generation of vancomycin resistance, the developmentof such resistance was neither easy nor recent.

Vancomycin resistance in enterococci is heterogeneous on many levels.Three phenotypes of vancomycin resistance (designated VanA, VanB, andVanC), each associated with a different ligase, are now well-described;a fourth, type VanD, has been recently reported. See, e.g., Noble, etal., 1992. Co-transfer of vancomycin and other resistance genes fromEnterococcus faecalis NCTC 12201 to Staphlococcus aureus. FEMSMicrobiol. Lett. 93: 195-198. VanA- and VanB-type resistance is encodedby gene clusters that are acquired (i.e., not part of the normal genomeof enterococci) and are often transferable. VanA-type strains aretypically highly resistant to vancomycin and moderately to highlyresistant to teicoplanin. This phenotype is often plasmid or transposonmediated and is inducible (i.e., exposure of bacteria to vancomycinresults in the induction of the synthesis of several proteins thattogether confer resistance). See, e.g., Hiramatsu, et al., 1997.Methicillin-resistant Staphylococcus aureus clinical strain with reducedvancomycin susceptibility. J. Antimicrob. Chemother. 40: 135-146. TheVanA gene cluster has been found in a small Tn3-like transposon, Tn1546,and in elements that appear to be closely related (e.g., Tn5488, whichhas an insertion sequence [IS 1251] within Tn1546. See, e.g.,Eliopoulos, et al., 1994. In vitro activities of two glycylcyclinesagainst gram-positive bacteria. Antimicrob. Agents Chemother. 38:534-541. These elements have, in turn, been found on both transferableand nontransferable plasmids, as well as on the chromosome of the hoststrain.

VanB type resistancewas initially not found to be transferable, but atleast in some instances, the VanBgene cluster has been found on large(i.e., 90 kb to 250 kb) chromosomally-located transferable elements, oneof which contains within it a 64-kb composite transposon (i.e., Tn1547).The VanB-cotnaining 64-kb transposon is part of a 250-kb mobile elementshown to move from the chromosome of one Enterococcus and insert intothe chromosome of another. Although not demonstrated, circularization ofthe vanB containing large mobile elements resembles the mechanismdescribed for conjugative transposons that can excise from thechromosome of one strain, circularize, transfer from one Enterococcus toanother, and reinsert into the chromosome of the recipient. The 64-kbtransposon can also jump to another plasmid within the host Enterococcusand that plasmid can then transfer by conjugation to other bacteria,taking the VanB resistance genes with it.

In contrast, VanC1 and VanC2 are normally occurring genes that areendogenous species characteristics of F. gaiinarum and F. casseliflavus,respectively, and are not transferable.

Therapeutic Approaches

Suitable antibiotics often are not available to treat MDR enterococcalinfections (e.g., endocarditis or bacteremia), in the presence ofneutropenia. Combinations of penicillin with vancomycin, ciprofloxacinwith ampicillin, or novobiocin with doxycycline, among others, have beenused, but can be unpredictable and remain clinically unproven. Thesubstantial drawback of the broad spectrum approach is that the moreorganisms affected (i.e., both protective commensals as well aspathogens), the more opportunities for resistance to evolve. Broadspectrum antibiotics permit empiric therapy in the absence of a specificdiagnosis and generate a more substantial return on investment in theshort-term. However, broad-spectrum antibiotics affect not onlydisease-causing organisms but also commensals present in numbers largeenough to generate resistance by otherwise rare mutational or geneticexchange events.

Although there are other therapeutic modalities under development (e.g.,targeted therapeutics), so long as the medical and pharmaceuticalcommunities continue to rely upon the use and development ofbroad-spectrum therapeutics as the principle therapeutic modality, acycle of drug introduction, followed by emergence of resistanceundoubtedly will continue.

With the current, dramatic increases in the number of bacterial strainswhich exhibit resistance to one or more antibiotics, the development ofa non-antibiotic-based therapeutic regimen is of paramount importance.Prior to the disclosure of the present invention, there remained a needfor the development of a highly efficacious biorational therapy whichfunctions therapeutically in acute treatment scenarios, as well asprophylactically and in vector control applications to mitigate or slowthe development of antibiotic-resistant pathogens (e.g.,antibiotic-resistant Enterococci) in both humans and animals, by thecolonization (or re-colonization) of the gastrointestinal tract withprobiotic microorganisms, which serves to reduce or prevent both thecolonization rate and the potential physiologically deleterious effectsdue to the colonization of antibiotic-resistant digestive pathogens.

In addition to enterococci, the probiotic composition of the presentinvention is effective against other common or antibiotic-resistantstrains of pathogens including, but not limited to, Candida,Clostridium, Escherichia, Klebsiella, Campylobacter, Peptococcus,Heliobacter, Hemophylus, Staphylococcus, Yersinia, Vibrio, Shigella,Salmonella, Streptococcus, Proteus, Pseudomonas, Toxoplasmosis, andRotovirus species. The advantages of such a non-antibiotic, probioticbacteria-based therapeutic regimen include, but are not limited to: (i)the administration of the composition will result reduction of thecolonization rate of enterococci in the gastrointestinal tract; (ii) nocontribution to the development of antibiotic resistance; (iii) thecomposition can be used prophylactically to reduce the reservoir ofenterococci in hospitals, which will concomitantly reduce the chances ofhigh-risk patients from acquiring VRE; (iv) the dosage of thecomposition can be varied according to patient age, condition, etc; and(v) the composition may be utilized in a food animal to reduce thedevelopment of further antibiotic resistance.

In an additional embodiment, skin creams, lotions, gels, and the like,which contain the novel stains of Bacillus coagulans disclosed herein,and/or the extracellular products thereof, would be effective in themitigation or prevention of pathogenic organisms on the skin, mucusmembrane, and cuticular tissues and further reduce the emergence ofantibiotic resistant pathogens. By way of example, and not oflimitation, the cells, spores, and/or extracellular products from thesenovel Bacillus coagulans strains could be incorporated into these skinproducts for this express purpose. For example, pathogenicantibiotic-resistant strains of Pseudomonas, Staphylococcus, and/orEnterococcus are frequently associated with infections of severe burns.Accordingly, the salves, lotions, gels, and the like, combined with thenovel Bacillus coagulans strains, and/or their extracellular products,as disclosed in the present invention, would be effective in mitigatingor preventing these pathogenic organisms. Additionally, administrationof these probiotic bacteria would help to achieve a state of properbiodiversity to the skin in burn cases, as, generally, such biodiversityis not associated with pathogenic overgrowth.

Probiotic, Lactic Acid-Producing Bacterial Strains

As utilized herein, “probiotic” refers to microorganisms that form atleast a part of the transient or endogenous flora and thereby exhibit abeneficial prophylactic and/or therapeutic effect on the host organism.Probiotics are generally known to be clinically safe (i.e.,non-pathogenic) by those individuals skilled in the art. By way ofexample, and not of limitation to any particular mechanism, theprophylactic and/or therapeutic effect of an acid-producing bacteria ofthe present invention results, in part, from a competitive inhibition ofthe growth of pathogens due to: (i) their superior colonizationabilities; (ii) parasitism of undesirable microorganisms; (iii) theproduction of acid (e.g., lactic, acetic, and other acidic compounds)and/or other extracellular products possessing anti-microbial activity;and (iv) various combinations thereof. It should be noted that theaforementioned products and activities of the acid-producing bacteria ofthe present invention act synergistically to produce the beneficialprobiotic effect disclosed herein.

A probiotic bacteria which is suitable for use in the methods andcompositions of the present invention: (i) possesses the ability toproduce and excrete acidic compounds (e.g., lactic acid, acetic acid,etc.); (ii) demonstrates beneficial function within the gastrointestinaltract; and (iii) is non-pathogenic. By way of example and not oflimitation, many suitable bacteria have been identified and aredescribed herein, although it should be noted that the present inventionis not to be limited to currently-classified bacterial species insofaras the purposes and objectives as disclosed. The physiochemical resultsfrom the in vivo production of lactic acid is key to the effectivenessof the probiotic lactic acid-producing bacteria of the presentinvention. Lactic acid production markedly decreases the pH (i.e.,increases acidity) within the local micro-floral environment and doesnot contribute to the growth of many undesirable,physiologically-deleterious bacteria and fungi. Thus, by the mechanismof lactic acid production, the probiotic inhibits growth of competingpathogenic bacteria.

Typical lactic acid-producing bacteria useful as a probiotic of thisinvention are efficient lactic acid producers, which includenon-pathogenic members of the Bacillus genus which produce bacteriocinsor other compounds which inhibit the growth of pathogenic organisms.

The Bacillus species, particularly those species having the ability toform spores (e.g., Bacillus coagulans), are a preferred embodiment ofthe present invention. The ability to sporulate makes these bacterialspecies relatively resistant to heat and other conditions, provides fora long shelf-life in product formulations, and is deal for survival andcolonization of tissues under conditions of pH, salinity, and the likewithin the gastrointestinal tract. Moreover, additional usefulproperties of many Bacillus species include being non-pathogenic,aerobic, facultative and heterotrophic, thus rendering these bacterialspecies safe and able to readily colonize the gastrointestinal tract.

Preferred methods and compositions disclosed herein utilize novelstrains of Bacillus coagulans and/or extracellular products thereof as aprobiotic. Prior to the invention, it was generally accepted that thevarious “classic” Lactobacillus and/or Bifidiobacterium species areunsuitable for colonization of the gut due to their instability in thehighly acidic environment of the gastrointestinal tract, particularlythe human gastrointestinal tract. The purified Bacillus coagulansstrains of the present invention are able to survive and colonize thegastrointestinal tract because the optimal temperature for growth islower than standard known strains of Bacillus coagulans. Additionally,probiotic Bacillus coagulans is non-pathogenic and is generally regardedas safe (i.e., GRAS classification) by the U.S. Federal DrugAdministration (FDA) and the U.S. Department of Agriculture (USDA), andby those individuals skilled within the art.

Because Bacillus coagulans possesses the ability to produceheat-resistant spores, it is particularly useful for makingpharmaceutical compositions, which require heat and pressure in theirmanufacture. Accordingly, formulations that include the utilizationviable Bacillus coagulans spores in a pharmaceutically-acceptablecarrier are particularly preferred for making and using compositionsdisclosed in the present invention.

The growth of these various Bacillus species to form cell cultures, cellpastes, and spore preparations is generally well-known within the art.Additionally, the present invention discloses methods for the isolationand partial purification of the extracellular products produced bycultures of Bacillus coagulans.

Commercial Sources of Traditional Strains of Bacillus coagulans

The Gram positive rods of Bacillus coagulans have a cell diameter ofgreater than 1.0 μm with variable swelling of the sporangium, withoutparasporal crystal production. Bacillus coagulans is a non-pathogenic,Gram positive, spore-forming bacteria that produces L(+) lactic acid(dextrorotatory) under homo-fermentation conditions. It has beenisolated from natural sources, such as heat-treated soil samplesinoculated into nutrient medium (see e.g., Bergey's Manual of SystemicBacteriology, Vol. 2, Sneath, P. H. A. et al., eds., Williams & Wilkins,Baltimore, Md., 1986). Purified Bacillus coagulans strains have servedas a source of enzymes including endonucleases (e.g., U.S. Pat. No.5,200,336); amylase (U.S. Pat. No. 4,980,180); lactase (U.S. Pat. No.4,323,651) and cyclo-malto-dextrin glucano-transferase (U.S. Pat. No.5,102,800). Bacillus coagulans has also been utilized to produce lacticacid (U.S. Pat. No. 5,079,164). A strain of Bacillus coagulans (alsoreferred to as Lactobacillus sporogenes; Sakaguti & Nakayama, ATCC No.31284) has been combined with other lactic acid producing bacteria andBacillus natto to produce a fermented food product from steamed soybeans(U.S. Pat. No. 4,110,477). Bacillus coagulans strains have also beenused as animal feeds additives for poultry and livestock to reducedisease and improve feed utilization and, therefore, to increase growthrate in the animals (International PCT Pat. Applications No. WO 9314187and No. WO 9411492). In particular, Bacillus coagulans strains have beenused as general nutritional supplements and agents to controlconstipation and diarrhea in humans and animals.

Bacillus coagulans cultures have been deposited with the followingprimary international culture collections: Agricultural Research ServiceCulture Collection; Russian Collection of Microorganisms; DeutscheSammlung von Mikroorganismen and Zellkulturen GmbH (German Collection ofMicroorganisms and Cell Cultures, VKM DSMZ); American Type CultureCollection (ATCC); Finnish Microorganism Collection (University ofGoteborg, Sweden); Japan Collection of Microorganisms (JCM); and JapanFederation for Culture Collection.

From the aforementioned deposits there are a total of eight lacticacid-producing bacterial species which have either been: (i) classifiedand deposited as Bacillus coagulans in the past but, have beenre-classified as another related Bacillus species; or (ii) deposited asanother closely related species but, have recently been re-classified asBacillus coagulans. These related species include, but are not limitedto, Bacillus coagulans, Bacillus stereothermophilus, Bacillusthermoacidurans, Lactobacillus sporogenes, Bacillus smithii, Bacillusdextrolacticus, Lactobacillus cereale, and Bacillus recemilacticus.However, there is currently some degree of confusion with respect to theclassification of these related bacterial strains—as there are no setrules for optimum, or even appropriate growth parameters, even betweensimilar strains. For example, Bacillus stereothermophilus is a Bacillusstrain known to have an optimum growth of approximately 55° C.

Various Bacillus coagulans bacterial strains which are currentlycommercially available from the American Type Culture Collection (ATCC,Rockville, Md.) include the following accession numbers: Bacilluscoagulans Hammer NRS 727 (ATCC No. 11014); Bacillus coagulans Hammerstrain C (ATCC No. 11369); Bacillus coagulans Hammer (ATCC No. 31284);and Bacillus coagulans Hammer NCA 4259 (ATCC No. 15949). PurifiedBacillus coagulans bacteria are also available from the DeutscheSarumlung von Mikroorganismen and Zellkuturen GmbH (Braunschweig,Germany) using the following accession numbers: Bacillus coagulansHammer 1915 (DSM No. 2356); Bacillus coagulans Hammer 1915 (DSM No.2383, corresponds to ATCC No. 11014); Bacillus coagulans Hammer (DSM No.2384, corresponds to ATCC No. 11369); and Bacillus coagulans Hammer (DSMNo. 2385, corresponds to ATCC No. 15949). Bacillus coagulans bacteriacan also be obtained from commercial suppliers such as SabinsaCorporation (Piscataway, N.J.) or K.K. Fermentation (Kyoto, Japan).

These aforementioned Bacillus coagulans strains and their growthrequirements have been described previously (see e.g., Baker, D. et al,1960. Can. J. Microbiol. 6: 557-563; Nakamura, H. et al, 1988. Int. J.Syst. Bacteriol. 38: 63-73. In addition, various strains of Bacilluscoagulans can also be isolated from natural sources (e.g., heat-treatedsoil samples) using well-known procedures (see e.g., Bergey's Manual ofSystemic Bacteriology, Vol. 2, p. 1117, Sneath, P. H. A. et al., eds.,Williams & Wilkins, Baltimore, Md., 1986).

Bacillus coagulans had originally been mis-characterized as aLactobacillus in view of the fact that, as originally described, thisbacterium was labeled as Lactobacillus sporogen{grave over (s)} (See,Nakamura et al. 1988. Int. J. Syst. Bacteriol. 38: 63-73). However,initial classification was incorrect due to the fact that Bacilluscoagulans produces spores and through metabolism excretes L(+)-lacticacid, both aspects which provide key features to its utility. Instead,these developmental and metabolic aspects required that the bacterium beclassified as a lactic acid Bacillus, and therefore it wasre-designated.

Biochemical Characteristics of Bacillus coagulans

Bacillus coagulans, being a member of the Bacillus genus, isspore-forming which upon activation in the acidic environment of thestomach, can germinate and proliferate in the intestine, produce thefavored L(+) optical isomer of lactic acid, and effectively prevent thegrowth of numerous bacterial and fungal pathogens. Table 1, below, is acomparative chart showing the biochemical attributes of lacticacid-producing bacteria and their similarities.

TABLE 1 Spirolacto- Bacillus Bacillus Lactobacillus bacillus PropertySpecies coagulans Species Species Catalase + + − − Benzidine + N/A − −Nitrate Red + N/A − − Gram Reaction + + + + Endospores + + − +Motility + +  −^(a) + Lactic Acid  −^(b) + + + m-A₂PM^(c) + +  −^(a) +Fatty Acid Bacillus- Lactobacillus- Variable Undefined Type Type ^(a)Lactobacillus plantarum may be motile and contains m-A₂ PMc in its cellwall ^(b)Some species including Bacillus coagulans can produce lacticacid ^(c)Meso-diaminopimelic acid ^(d)Data not Available

Known Lactobacillus species are generally believed to be unsuitable forcolonization of the gut due to their instability in the harsh (i.e.,acidic) pH environment of the digestive tract, e.g., in the presence ofbile, particularly human bile. This instability is one of the primaryreasons why the use of lactic acid-producing bacterial strains asprobiotics has not been more vigorously explored.

In contrast to these aforementioned bacterial species, Bacilluscoagulans is able to survive, colonize, and grow in the gastrointestinaltract. In particular, the human bile environment is different from thebile environment of animal models, and growth of Bacillus coagulans inhuman gastrointestinal tract models has not been described. Thefollowing proliferative attributes illustrate the strengths of Bacilluscoagulans over other species of lactic acid-producing bacteria include,but are not limited to:

Facultative Aerobe:

-   -   Bacillus coagulans possesses the ability to grow well in either        environments that have free-oxygen or in strictly anaerobic        environments. This is important due to the fact that        Lactobacilli and Bifidobacteria are not aero-tolerant. Thus, in        essence, these aforementioned bacterial species are strictly        anaerobic and do not proliferate well in environments containing        free-oxygen. Because Bacillus coagulans is viable in a        free-oxygen environment, it can be used in surface-active        formulations (e.g., skin powders, creams, ointments, etc) to act        prophylactically against the overgrowth of pathogens.

Thermo-Tolerant:

-   -   The vegetative cells of Bacillus coagulans possess the ability        to grow at temperatures as high as 65° C., whereas the        endospores can withstand temperatures in excess of 100° C. In        fact, Bacillus coagulans, along with Bacillus        stereothermophilus, is used for quality control purposes in        autoclaves. This fact is crucial due to the frailty of all        Lactobacilli and Bifidobacteria. For a bacterium to have        commercial viability it must be stabile and viable at the time        of packaging. This viability must be retained in order to        deliver an efficacious product to the consumer.

Halo-Tolerant:

-   -   Bacillus coagulans possesses the ability to grow in highly        alkaline environments including 7% NaCl or 10% caustic soda.

The characteristics of Bacillus coagulans, as cited in Bergey's Manual(Seventh Edition), include: Gram-positive spore-forming rodsapproximately 0.9 μm×3.0-5.0 μm in size; aerobic to microaerophilic;produce L(+) (dextrorotatory) lactic acid in a homofermentative manner.Due to the fact that Bacillus coagulans exhibits characteristics typicalof both genera Lactobacillus and Bacillus, its taxonomic positionbetween the families Lactobacillaceae and Bacillaceae has often beendiscussed.

It is often very difficult to distinguish between two species ofbacteria, which are morphologically similar and possess similarphysiological and biochemical characteristics. DNA homology analysis isa useful technique in resolving this difficulty. The base composition(i.e., % GC content) and the specific nucleotide sequence of thebacterial DNA generally differs between bacterial species andsub-species. Additionally, DNA from closely related bacteria hybridizewith each other more efficiently. It the present invention, theseaforementioned methodologies have been effectively employed todifferentiate, as well as to recognize the innate resemblance betweenBacillus coagulans and members of the genus Lactobacillus and tovalidate it's taxonomical placement under genus Bacillus.

Table 2, below, discusses the colony morphology of Bacillus coagulans.

TABLE 2 Cells are long and slender (0.3 to 0.8 μm), some are bent andall the cells have rounded ends Motile with peritrichous flagellas. Grampositive. Colonies are usually 2.5 mm in diameter, convex, smooth,glistening and do not produce any pigment. Extremely fastidiousorganisms requiring complex organic substrates for growth such asfermentable carbohydrate, peptone, meat and yeast extract. MRS mediumsupplemented with tomato juice, manganese, acetate and Tween-80 is asuitable medium for growth. Grow optimally at 40° C. to 50° C. and theoptimum pH in the range 5.5 to 6.2. Micro-aerophilic, exhibitfermentative metabolism and are facultatively aerobic. Produce acid fromarabinose, xylose, glucose, galactose, mannose, fructose, maltose,sucrose, and trehalose. Do not hydrolyze starch or casein. Do notliquefy gelatin. Are indole negative and do not produce hydrogen sulfideor gas. Produce L (+) (dextrorotatory) lactic acid from glucose,fructose, sucrose, trehalose. Menaquinones are absent.Table 3, below, discusses the mechanism of carbohydrate fermentationutilized by Bacillus coagulans:

TABLE 3 Carbohydrate Acid Production Gas Production Inulin − − Maltose +− Mannitol + − Raffinose + − Sorbitol − − Sucrose + − Trehalose + −4. Biological “Safety” of Bacillus coagulans

Bacillus coagulans enjoys a longer safe history of use than most of thecommon Lactobacillus and Bifidobacterium species that are commonly soldas “nutritional supplements” at health food stores, or used in theproduction of cultured dairy products.

General recognition of biological safety may be based only upon theviews of experts qualified by scientific training and experience toevaluate the safety of substances directly or indirectly added to food.The basis of such views may be derived through:

-   -   (1) Scientific procedures.    -   (2) In the case of a substance used in food prior to Jan. 1,        1958, through experience based on common use in food. General        recognition of safety requires common knowledge about the        substance throughout the scientific community knowledgeable        about the safety of substances directly or indirectly added to        food.    -   (3) General recognition of safety based upon scientific        procedures shall require the same quantity and quality of        scientific evidence as is required to obtain approval of a food        additive regulation for the ingredient. General recognition of        safety through scientific procedures shall ordinarily be based        upon published studies, which may be corroborated by unpublished        studies and other data and information.    -   (4) General recognition of safety through experience based on        common use in food prior to Jan. 1, 1958, may be determined        without the quantity or quality of scientific procedures        required for approval of a food additive regulation. General        recognition of safety through experience based on common use in        food prior to Jan. 1, 1958, shall be based solely on food use of        the substance prior to Jan. 1, 1958, and shall ordinarily be        based upon generally available data and information.

Lactic acid-producing bacteria are a necessary component in fermenteddairy products. Due to the fact that Bacillus coagulans was firstisolated in 1932, has been used in the production of food products priorto Jan. 1, 1958, and has not been implicated in any pathogenic oropportunistic diseases since its isolation, it qualifies under as manyas 9 sections and subsections of the United States Federal Registry forGRAS (Generally-Regarded as Safe) listing. The GRAS list simplyindicates that a food additive is not thought to illicit any toxigenicor pathogenic response and is considered safe by those skilled in theart of food science, biochemistry, and microbiology.

Bacillus coagulans, subspecies Hammer (ATCC-31284), was first isolatedas a soil isolate at Yamanashi University in 1933 by Nakayama. Bacilluscoagulans species are usually soil isolate. With the exception ofBacillus cereus and Bacillus anthraices, Bacillus species are known tobe benign in the environment. To date, there have been no references ofany species of Bacillus coagulans being involved in a pathogenic oropportunistic illness. Similarly, in an analysis of published data,there have also been no clinical trials that had been compromised due topathogenesis by lactic acid-producing bacteria. In view of these facts,which are not disputed within the relevant scientific fields, Bacilluscoagulans is safe as a therapeutic compositions.

Sensitivity of Bacillus coagulans to Antibiotics

Although GRAS-listed organisms are safe for use in “normal”,immunocompetent individuals, susceptible individuals (e.g.,immunosuppressed, immunocompromised, organ transplant, etc.) may be atrisk to develop bacteremia or septicemia through the ingestion ofbacterial products that are thought to be biologically safe. Althoughthere have been peer-reviewed articles that have shown Lactobacilli tobe implicated in severe systemic infections (i.e., opportunisticpathogenesis), there have been no reports which have shown a Bacilluscoagulans-mediated etiology. Notwithstanding the foregoing, studies arecurrently underway in immunocompromised mice/rats to determine whetherthese novel strains of Bacillus coagulans have any potential for suchopportunistic pathogenesis.

Analysis of the antibiotic sensitivity of Bacillus coagulans, subspeciesHammer (ATCC-31284) was performed using the Kirby-Bauer (countingcolonies on plates) and Vitek (optical density of culture)susceptibility testing methodologies in order to ascertain the specificantibiotic compound(s) that would be effective in eliminating a Bacilluscoagulans colonization, if needed, regardless of rational. UsingKirby-Bauer testing, Bacillus coagulans was found to be susceptible to:ampicillin; ciprofloxacin; trimethoprim-sulfamethoxazole; rifampin;erythromycin; vancomycin; gentamicin; oxacillin, and possessedintermediate susceptibility to tetracycline. Using Vitek testing,Bacillus coagulans was found to be susceptible to: penicillin;vancomycin; gentamicin (500 μg/ml); streptomycin (2,000 μg/ml);nitrofurantoin; norfloxacin; chloramphenicol, and was resistant totetracycline. Additionally, Nitrocefin testing was performed andindicated Bacillus coagulans was positive for low-level β-lactamaseproduction.

Production of Anti-Microbial Substances by Bacillus coagulans

Bacteriocins are proteins or protein-particulate complexes withbactericidal activities directed against species, which are closelyrelated to the producer bacterium. The inhibitory activity of lacticacid-producing bacteria (e.g., Bacillus coagulans) towards putrefactiveorganisms is thought to be partially due to the production ofbacteriocins.

Table 4, below, lists some of the various bacterocins, which have beenisolated and characterized from lactic acid-producing bacterial species.

TABLE 4 Bacterocin Bacterial Species Acidolin Lactobacillus acidophilusAdidophilin Lactobacillus acidophilus Lactacin B Lactobacillusacidophilus Lactacin F Lactobacillus acidophilus Bulgarin Lactobacillusbulgaricus Plantaracin SIK-83 Lactobacillus plantarum Plantaracin ALactobacillus plantarum Lactolin 27 Lactobacillus helveticus HelveticinJ Lactobacillus helveticus Reuterin Lactobacillus reuteri LactobrevinLactobacillus brevis Lactobacillin Lactobacillus brevis

Additionally, lactic acid-producing bacteria also inhibit the growth ofpathogenic/putrefactive microorganisms through other metabolic productssuch as hydrogen peroxide, carbon dioxide, and diacetyl.

The metabolites of lactic acid-producing bacteria that exertantagonistic actions against pathogenic bacteria are summarized below inTable 5.

TABLE 5 Metabolic Product Mode of Antagonistic Action Carbon DioxideInhibits decarboxylation Reduces membrane permeability DiacetylInteracts with arginine-binding proteins Hydrogen peroxide/ Oxidizesbasic proteins Lactoperoxidase Lactic Acid Undisssociated lactic acidpenetrates the membranes, lowering the intracellular pH. Interferes withmetabolic processes such as oxidative phosphorylation. BacterocinsAffects membranes, membrane-associated replication and DNA/proteinsynthesis.

The levels of optical isomeric forms of lactic acid produced depend uponthe specific species of the bacterium. The structural configurations ofthese isomers are as follows:

In humans, both isomers are absorbed from the intestinal tract. WhereasL(+) lactic acid is completely and rapidly metabolized in glycogensynthesis, D(−) lactic acid is metabolized at a lesser rate and thenon-metabolized acid is excreted in the urine. The presence ofun-metabolized lactic acid results in metabolic acidosis in infants.Lactobacillus acidophilus produces the D(−) form and is therefore ofdisputable clinical benefit. In contrast, Bacillus coagulans, producesonly L(+) lactic acid, and hence is preferred over other species oflactic acid-producing bacteria which produce the D(−) form.

Purified Novel Strains of Bacillus coagulans

Previously-available strains of lactic acid-producing bacteria(including Bacillus coagulans ATCC-type stain #31284) were ineffectualas probiotics due to various factors including, but not limited to,their high optimal growth temperature (i.e., >40° C.) requirement andtheir requirement for an 80° C. “spore shock” for spore germination.These requirements were incompatible with the use of thesepreviously-available strains of Bacillus coagulans as probiotics, intherapeutic compositions (e.g., in the treatment of antibiotic-resistantgastrointestinal pathogens), and the like.

Bacillus coagulans described herein possess biochemical andphysiological characteristics which include, but are not limited to: (i)the production of the (L)+ optical isomer of lactic acid (propionicacid); (ii) have an optimal growth temperature of less than 45° C.;(iii) the production of spores resistant to temperatures of up toapproximately 90° C. which are able to germinate in a human or animalbody without specific inducement (e.g., spore-shock or otherenvironmental factors); (iv) the production of one or more extracellularproducts exhibiting probiotic activity which inhibits the growth ofbacteria, yeast, fungi, virus, or any combinations thereof; and/or (v)the ability to utilize a wide spectrum of substrates for proliferation.These novel strains will be more fully discussed, below.

Disclosed herein are three previously uncharacterized strains ofBacillus coagulans which have markedly lower growth temperature optima,while still possessing the ability to produce lactic acid and otherextracellular products under laboratory fermentation conditions. Thesenovel strains share some characteristics with ATCC-type strain(ATCC-31284), but they possess differences which, e.g., lower growthtemperature optima, which increase their efficacy for use as probiotics.

These novel strains were originally discovered in a mixed microbialcommunity of Bacillus coagulans colonies where they exhibiteddifferences in both colony morphology and optimal growth temperaturefrom that of the Bacillus coagulans ATCC-type strain (hereinafter“ATCC-31284” or “ATCC-99%”). These novel strains are characterized asfollows:

Bacillus coagulans 1% isolate-designated GBI-1

Bacillus coagulans 20° C. isolate-designated GBI-20

Bacillus coagulans 30° C. isolate-designated GBI-30

Bacillus coagulans 40° C. isolate-designated GBI-40

These novel strains of Bacillus coagulans 1% isolate (hereinafterdesignated “GBI-1”); the Bacillus coagulans strain which possesses aoptimal growth temperature of 20° C. (hereinafter designated “GBI-20);the Bacillus coagulans strain which possesses a optimal growthtemperature of 30° C. (hereinafter designated “GBI-30); and the Bacilluscoagulans strain which possesses a optima growth temperature of 40° C.(hereinafter designated “GBI-40). The biochemical, physiological, andmorphological characteristics of these novel strains of Bacilluscoagulans will be fully discussed in the Specific Examples section,infra.

Treatment of Antibiotic-Resistant Bacterial Gastrointestinal Infections

The present invention contemplates a method for treating, reducing orcontrolling antibiotic-resistant bacterial gastrointestinal infectionsusing the therapeutic composition or therapeutic system disclosedherein. The disclosed methods of treatment function so as to inhibit thegrowth of the pathogenic bacteria which are associated withgastrointestinal infections, as well as to concomitantly mitigate thedeleterious physiological effects/symptoms of these pathogenicinfections.

The novel strains of Bacillus coagulans disclosed herein are generallyregarded as safe by those skilled within the art (i.e., GRAS Certifiedby the FDA) and, therefore, suitable for direct ingestion in food stuffsor as a food supplement. The methods of the present invention compriseadministration of a therapeutic composition containing one or moreBacillus coagulans strains and/or the extracellular products thereof, tothe gastrointestinal tract of a human or animal, to treat or preventbacterial infection. Administration is preferably made using a liquid,powder, solid food and the like formulation compatible with oraladministration, all formulated to contain a therapeutic composition ofthe present invention by use of methods well-known within the art.

The methods of the present invention includes administration of acomposition containing one or more of the following: Bacillus coagulansbacterial cells (i.e., vegetative bacterial cells); spores; and/orisolated Bacillus coagulans extracellular products (which contains ametabolite possessing antibiotic-like properties) to a human or animal,so as to treat or prevent the colonization of antibiotic-resistantpathogens with the gastrointestinal tract. In particular, for VRE, VISA,PRP, and other pathogens, the methods includes administering to thepatient, for example, Bacillus coagulans in food or as a foodsupplement. Oral administration is preferably in an aqueous suspension,emulsion, powder or solid, either already formulated into a food, or asa composition which is added to food by the user prior to consumption.Administration to the gastrointestinal tract may also be in the form ofan anal suppository (e.g., in a gel or semi-solid formulation). All suchformulations are made using standard methodologies.

Administration of a therapeutic composition is preferably to thegastrointestinal tract using a gel, suspension, aerosol spray, capsule,tablet, powder or semi-solid formulation (e.g., a suppository)containing a therapeutic composition of the present invention, allformulated using methods well-known within the art. Administration ofthe compositions containing the active probiotic lactic acid-producingbacterium which is effective in preventing or treating a pathogenicbacterial infection, generally consist of one to ten dosages ofapproximately 10 mg to 10 g of the therapeutic composition per dosage,for a time period ranging from one day to one month. Administrations are(generally) once every twelve hours and up to once every four hours. Inthe preferred embodiment, two to four administrations of the therapeuticcomposition per day, of approximately 0.1 g to 5 g per dose, for one toseven days. This preferred dose is sufficient to prevent or treat apathogenic bacterial infection. Of course, the specific route, dosageand timing of the administration will depend, in part, upon theparticular pathogen and/or condition being treated, as well as theextent of said condition.

An embodiment of the present invention involves the administration offrom approximately 1×10³ to 1×10¹⁴ CFU of viable, vegetative bacteria orspore per day, more preferably from approximately 1×10⁵ to 1×10¹⁰, andmost preferably from approximately 5×10⁸ to 1×10⁹ CFU of viable,vegetative bacteria or spores per day. Where the condition to be treatedinvolves antibiotic-resistant digestive pathogens and the patient is anadult, the typical dosage is approximately 1×10² to 1×10¹⁴ CFU ofviable, vegetative bacteria or spores per day, preferably fromapproximately 1×10⁸ to 1×10¹⁰, and more preferably from approximately2.5×10⁸ to 1×10¹⁰ CFU of viable, vegetative bacteria or spores per day.

Another embodiment of the present invention discloses the administrationof a composition comprising a total concentration ratio of Bacilluscoagulans extracellular product ranging from approximately 1% to 90%extracellular product with the remainder comprising the carrier ordelivery component. A preferred embodiment comprises a composition atotal concentration ratio of Bacillus coagulans extracellular productranging from approximately 10% to 75% extracellular product with theremainder comprising the carrier or delivery component.

The present invention further contemplates a therapeutic system fortreating, reducing and/or controlling pathogenic bacterial infections.Typically, the system is in the form of a package containing atherapeutic composition of the present invention, or in combination withpackaging material. The packaging material includes a label orinstructions for use of the components of the package. The instructionsindicate the contemplated use of the packaged component as describedherein for the methods or compositions of the invention.

By way of example, and not of limitation, a system can comprise one ormore unit dosages of a therapeutic composition according to the presentinvention. Alternatively, the system can alternately contain bulkquantities of a therapeutic composition. The label contains instructionsfor using the therapeutic composition in either unit dose or in bulkforms as appropriate, and may also include information regarding storageof the composition, disease indications, dosages, routes and modes ofadministration and the like information.

Furthermore, depending upon the particular contemplated use, the systemmay optionally contain either combined or in separate packages one ormore of the following components: bifidogenic oligosaccharides,flavorings, carriers, and the like components. One particularlypreferred embodiment comprises unit dose packages of Bacillus coagulansspores for use in combination with a conventional liquid product,together with instructions for combining the probiotic with the formulafor use in a therapeutic method.

Inhibition of Pathogens and Parasites in Animals

The present invention also discloses compositions and methods of use forinhibiting growth of parasites and/or antibiotic-resistant pathogenicorganisms in the gastrointestinal tract of animals. As used herein, theterms “pathogen” and “parasite” are used interchangeably in the contextof a deleterious organism growing in the gastrointestinal tract and/orfeces of an animal, although it appreciated that these terms havedistinctive meanings.

The present invention describes compositions and methods of use forinhibiting or preventing growth of a pathogen in the gastrointestinaltract of an animal comprising the step of administering a composition ofthe invention to the gastrointestinal tract of the animal one or more ofthe following: Bacillus coagulans bacterial cells (i.e., vegetativebacterial cells); spores; and/or isolated Bacillus coagulansextracellular products (which contains a metabolite possessingantibiotic-like properties) to the animal, so as to treat or prevent thecolonization of antibiotic-resistant pathogens with the gastrointestinaltract. In particular, for VRE, VISA, PRP, and other pathogens, themethods includes administering to the animal, for example, Bacilluscoagulans in food or as a food supplement. Oral administration ispreferably in an aqueous suspension, emulsion, powder or solid, eitheralready formulated into a food, or as a composition which is added tofood by the user prior to consumption. Administration to thegastrointestinal tract may also be in the form of an anal suppository(e.g., in a gel or semi-solid formulation). All such formulations aremade using standard methodologies.

The method comprises administration of a composition of this inventioncontaining the active ingredients to an animal in various dosageregimens as described herein to achieve the nutritional result.Administration of the compositions containing the active ingredientseffective in inhibiting parasite growth in the intestine and in fecesgenerally consist of one to ten unit dosages of 10 mg to 10 g per dosageof the composition for one day up to one month for an animal ofapproximately 100 kg body weight. Unit dosages are generally given onceevery twelve hours and up to once every four hours. Preferably two tofour dosages of the composition per day, each comprising about 0.1 g to50 g per dosage, for one to seven days are sufficient to achieve thedesired result.

A preferred method involves the administration into the digestive tractof from 1×10² to 1×10¹⁰ viable bacterium or spore per day, in someembodiments from 1×10³ to 1×10⁶, in other embodiments from 1×10⁶ to1×10⁹, and more preferably about from 5×10⁸ to 1×10⁹ viable bacterium orspore per day. Exemplary dosages range from about 1×10³ to 1×10⁶ viablebacterium per day, or alternatively range from about 1×10⁶ to 1×10⁹viable bacterium per day.

Another embodiment of the present invention discloses the administrationof a composition comprising a total concentration ratio of Bacilluscoagulans extracellular product ranging from approximately 1% to 90%extracellular product with the remainder comprising the carrier ordelivery component. A preferred embodiment comprises a composition atotal concentration ratio of Bacillus coagulans extracellular productranging from approximately 10% to 75% extracellular product with theremainder comprising the carrier or delivery component.

The method is typically practiced on any animal where inhibitingpathogen or parasites is desired. The animal can be any livestock orzoological specimen where such inhibition of parasites/pathogensprovides economic and health benefits. Any animal can benefit by theclaimed methods, including birds, reptiles, mammals such as horses,cows, sheep, goats, pigs, and the like domesticated animals, or any of avariety of animals of zoological interest. Other purposes are readilyapparent to one skilled in the arts of nutrient absorption, feedutilization and bioavailability.

The present invention further contemplates a therapeutic system fortreating, reducing and/or controlling pathogenic bacterial infections.Typically, the system is in the form of a package containing atherapeutic composition of the present invention, or in combination withpackaging material. The packaging material includes a label orinstructions for use of the components of the package. The instructionsindicate the contemplated use of the packaged component as describedherein for the methods or compositions of the invention.

By way of example, and not of limitation, a system can comprise one ormore unit dosages of a therapeutic composition according to the presentinvention. Alternatively, the system can alternately contain bulkquantities of a therapeutic composition. The label contains instructionsfor using the therapeutic composition in either unit dose or in bulkforms as appropriate, and may also include information regarding storageof the composition, disease indications, dosages, routes and modes ofadministration and the like information.

Furthermore, depending upon the particular contemplated use, the systemmay optionally contain either combined or in separate packages one ormore of the following components: bifidogenic oligosaccharides,flavorings, carriers, and the like components. One particularlypreferred embodiment comprises unit dose packages of Bacillus coagulansspores for use in combination with a conventional liquid product,together with instructions for combining the probiotic with the formulafor use in a therapeutic method.

Insofar as feces provide growth and breeding grounds for undesirableorganisms, controlling and/or inhibiting growth of parasites andpathogenic organisms in feces inhibits growth and reproduction of theseundesirable organisms in areas where feces is produced, deposited and/orstored. For example, in barns or corrals, in animal cages, in feed lots,in zoological display enclosures, and the like areas where animals aremaintained and feces is deposited, there is an opportunity forparasites/pathogens to irritate, spread, reproduce and/or infect otherhosts. These circumstances provide a variety of undesirable problemssolved by the present invention. For example, it is undesirable forparasites or pathogens to spread and further infect hosts, and thereofor any means to control spread of infection is of great benefit wheremultiple animals are caged together. In addition, in many circumstancesbiting of host animals by parasites or flying insects irritates and/orupsets animals, providing behavior problems which includes excessivekicking, biting and related activities which are unsafe for neighboringanimals and for animal handlers.

In an another embodiment, the invention contemplates a method forreducing and/or controlling flying insect populations in animalcages/pens/enclosures where animals are maintained comprisingadministering a composition of the present invention to thegastrointestinal tract of the caged animals.

The present invention is useful at controlling a large variety ofparasites and pathogenic organisms, and therefore the invention need notbe limited to inhibiting any particular genus or species of organism.For example, based on the mechanisms described herein for effectivenessof the composition, it is seen that all insect varieties which can actas an animal parasite can be targeted by the methods of the presentinvention. Parasites can infect any of a variety of animals, includingmammals, reptiles, birds and the like, and therefore the invention isdeemed to not be limited to any particular animal. Examples ofwell-known or important parasites are described herein for illustrationof the invention, but are not to be viewed as limiting the invention.Representative parasites and animal and/or human hosts are described inextensive detail in a variety of veterinary treatises such as “Merck' sVeterinary Manual” and “Cecils' Human Diseases” Parasites of horsesincludes horse bots, lip bots or throat bots, caused by Gasterophilusspecies, such as G. intestinalis, G. haemorrhiodalis, and G. nasalis,stomach worms, caused by Habronema species, such as H. muscae or H.microstoma mulus, or caused by Crascia species, such as C. mepastoma, orcaused by Trichostrongvlus species, such as T. axei, ascarids (whiteworms) caused by Parascaris species such as P. eciuorum, blood worms(palisade worms, red worms or sclerostomes) caused by Stroncrvlusspecies such as S. vulcraris, S. epuinus or S. edentatus, smallstrongyles of the cecum and colon caused by Triodontophorus species suchas T. tenuicollis, pinworms caused by Oxvuris species such as O. eaui,strongyloides infections of the intestine caused by Stroncivloideswesteri, tapeworms caused by Anonlocephala species such as A. macma andA. perfoliata, and caused by Paranonlocephala mamillana.

Various other parasites cause disease in ruminants, typically cattle,include the wire worm (or barber's pole worm or large stomach worm)caused by Haemonchus species. Parasites caused in ruminants, typicallyswine, include stomach worms caused by Hvostroncmulus species.

Additional parasites are known to infect a variety of animal hosts, andtherefore are a target for treatment by the methods of the presentinvention. For example, gastrointestinal parasites infect a variety ofanimals and can include Spirocerca species such as S. lupi that causeesopheageal worms in canines and Physoloptera species that cause stomachworms in canines and felines.

Where the animal is fed a pelletized or granular food, the compositioncan be included in the pelletized or granular food, or can comprise amixture of the pelletized food combined with a pelletized composition ofthis invention. Mixing pelletized food with a pelletized formulation ofa composition of this invention is a particularly preferred method forpracticing the present invention, insofar as it provides a convenientsystem for using commercial feeds and simultaneously regulating theamounts of a composition of this invention to be administered.

Administration of a therapeutic composition is preferably to the gutusing a gel, suspension, aerosol spray, capsule, tablet, granule,pellet, wafer, powder or semi-solid formulation (e.g., a suppository)containing a nutritional composition of this invention, all formulatedusing methods well known in the art.

The present invention further contemplates a system for inhibitinggrowth of parasites and/or pathogens in the gastrointestinal tract of ananimal or in animal feces comprising a container comprising label and acomposition according to the present invention, wherein said labelcomprises instructions for use of the composition for inhibitingpathogen/parasite growth.

Typically, the system is present in the form of a package containing acomposition of this invention, or in combination with packagingmaterial. The packaging material includes a label or instructions foruse of the components of the package. The instructions indicate theontemplated use of the package component as described herein for themethods or compositions of the invention.

For example, a system can comprise one or more unit dosages of atherapeutic composition according to the invention. Alternatively, thesystem can contain bulk quantities of a composition. The label containsinstructions for using the composition in either unit dose or in bulkforms as appropriate, and may include information regarding storage ofthe composition, feeding instruction, health and diet indications,dosages, routes of administration, methods for blending the compositionwith pre-selected food stuffs, and the like information.

A. Culture of Bacillus coagulans

Bacillus coagulans is aerobic and facultative, and is typically culturedat pH 5.7 to 6.8, in a nutrient broth containing up to 2% (by wt) NaCl,although neither NaCl, nor KCl are required for growth. A pH ofapproximately 4.0 to 7.5, is optimum for initiation of sporulation(i.e., the formation of spores). The novel strains of Bacillus coagulansdisclosed herein are optimally grown at 20° C. to 40° C., and the sporescan withstand pasteurization. Additionally, the bacteria exhibitfacultative and heterotrophic growth by utilizing a nitrate or sulfatesource.

Bacillus coagulans can be cultured in a variety of media, although ithas been demonstrated that certain growth conditions are moreefficacious at producing a culture which yields a high level ofsporulation. For example, sporulation is demonstrated to be enhanced ifthe culture medium includes 10 mg/l of MgSO₄ sulfate, yielding a ratioof spores to vegetative cells of approximately 80:20. In addition,certain culture conditions produce a bacterial spore which contains aspectrum of metabolic enzymes particularly suited for the presentinvention (i.e., production of lactic acid and enzymes for the enhancedprobiotic activity and biodegradation). Although the spores produced bythese aforementioned culture conditions are preferred, various othercompatible culture conditions which produce viable Bacillus coagulansspores may be utilized in the practice of the present invention.

Suitable media for the culture of Bacillus coagulans include: PDB(potato dextrose broth); TSB (tryptic soy broth); and NB (nutrientbroth), which are all well-known within the field and available from avariety of sources. In one embodiment of the present invention, mediasupplements which contain enzymatic digests of poultry and/or fishtissue, and containing food yeast are particularly preferred. Apreferred media supplement produces a media containing at least 60%protein, approximately 20% complex carbohydrates, and approximately 6%lipids. Media can be obtained from a variety of commercial sources,notably DIFCO (Newark, N.J.); BBL (Cockeyesville, Md.); AdvancedMicrobial Systems (Shakopee, Minn.); and Troy Biologicals (Troy, Md.).An efficacious growth medium for Bacillus coagulans is a Glucose Yeastextract (GYE) medium. The formulation for GYE is shown below in Table 6.

TABLE 6 Yeast Extract Powder 5.0 grams Casitone (Peptone) 5.0 gramsD-glucose 3.0 grams K₂HPO₄ 0.5 grams KH₂PO₄ 0.5 grams MgSO₄ 0.3 gramsTrace Minerals Solution (see, Table 5) 1.0 ml Distilled Water 1000 mlAgar (to be added after pH adjustment) 15.0 GramsThe pH of the medium was then adjusted to approximately 6.3 followed bysterilization with steam at 1.2 kg/cm² pressure at 120° C. for 15minutes.

The formulation for the trace mineral solution utilized for the analysisof the Bacillus coagulans bacterial strain of the present invention isshown below in Table 7.

TABLE 7 NaCl 500 mg MnSO₄•5H₂O 500 mg ZnSO₄•7H₂O 80 mg CuSO₄•5H₂O 80 mgCoSO₄•7H₂O 80 mg Distilled Water 50 ml

(i) Small-Scale Culture

Small-scale culture of Bacillus coagulans may be accomplished by use ofthe aforementioned Glucose Yeast extract (GYE) medium. The medium wasinoculated and grown to a cell density of approximately 1×10⁸ to 1×10⁹cells/ml. The bacteria were cultured by utilization of a standardairlift fermentation vessel at 30° C. The range of MnSO₄ acceptable forsporulation was found to be 1.0 mg/l to 1.0 g/l. The vegetativebacterial cells can actively reproduce up to 65° C., and the spores arestable up to 90° C.

Following culture, the Bacillus coagulans bacterial cells or spores werecollected using standard methods (e.g., filtration, centrifugation) andthe collected cells and spores may subsequently be lyophilized, spraydried, air dried or frozen. As described herein, the supernatant fromthe cell culture can be collected and used as an extracellular agentsecreted by Bacillus coagulans which possesses anti-microbial activityuseful in a formulation of this invention.

A typical yield obtained from the aforementioned culture methodology isin the range of approximately 1×10⁹-1×10¹³ viable spores and, moretypically, approximately 10-15×10¹⁰ cells/spores per gram prior to beingdried. It should also be noted that the Bacillus coagulans spores,following a drying step, maintain at least 90% viability for up to 7years when stored at room temperature. Hence, the effective shelf-lifeof a composition containing Bacillus coagulans Hammer spores at roomtemperature is approximately 10 years.

(ii) Large-Scale Culture

Large-scale, batch fermentation of Bacillus coagulans may beaccomplished by use of the aforementioned Glucose Yeast extract (GYE)medium. The fermentation vessel may include: a 500 liter 314 seriesstainless airlift fermentation vessel with 60 psi pressure rating; Hannaduel set-point pH control system with in-process electrode; Highpressure turbine blower with 0.2 μm in-line filters for sterile airfeed; a 10 kw process temperature controller; and appropriate highburst-pressure stainless steel sanitary hose and fittings.

Batch fermentation comprises the following procedure. A single colony ofBacillus coagulans was selected with sterile loop from a petri-dishcolony. This single colony was then used to inoculate a two-literErlenmeyer flask containing GYE media, dextrose, and minerals. Theculture was incubated for approximately 18 hours in an orbital shaker(possessing a 2″ orbit) at 35° C. This 2 liter culture was used toinoculate a sterilized, 500 liter Batch Fermenter containing GYE media,dextrose, and minerals. The Batch Fermenter was run at 35° C. forapproximately 30 hours under high aeration (36-38 LPM). Following thisincubation, the Batch Fermenter aeration was turned off and temperaturereduced to 20° C. for 4 hours to facilitate settling of the bacterialcells therein. The fermentation broth was harvested using Alpha-LavalSharples continuous-feed centrifuge at 12,000 rpm at 10° C. and thebacterial solids were removed for subsequent lyophilization.

Preparation of Bacillus coagulans Spores

A culture of dried Bacillus coagulans spores may be prepared, e.g., asfollows. Approximately 1×10⁷ spores were inoculated into one liter ofculture medium containing: 30 g (wt./vol GYE media, dextrose, andminerals. The culture was maintained for 72 hours under a high oxygenenvironment at 37° C. so as to produce a culture having approximately15×10¹⁰ cells/gram of culture. The culture was then filtered to removethe liquid culture medium and the resulting bacterial pellet wasresuspended in water and lyophilized. The lyophilized bacteria wereground to a fine “powder” by use of standard good manufacturing practice(cGMP) methodologies.

Preparation of Bacillus coagulans Extracellular Products

A culture of Bacillus coagulans was prepared as described in SpecificExample A(i)-(ii). The culture was maintained for 5 days as described.The culture was first autoclaved for 30 minutesat 250° F., and thencentrifuged at 4000 r.p.m. for 15 mm. The resulting supernatant wascollected and subjected to sub-micron filtration by the initial use of aBuchner funnel with a 0.8 μm filter. The filtrate was then collected andfurther filtered through a 0.2 μm Nalge vacuum filter. The resultingfiltrate was then collected (an approximate volume of 900 ml/liter ofculture medium) and comprised a liquid containing an extracellularproduct, which was to be quantitatively analyzed and utilized in thesubsequent inhibition studies.

The following methodologies were utilized to characterize thesupernatant.

Liquid Chromatography of Proteins:

20 ml of culture supernatant was loaded on an analytical Mono 9chromatography column (Pharmacia) equilibrated in Buffer A (0.25 M TrisHCl; pH 8.0) using a BioCAD Sprint chromatography system (PerseptiveBiosystems, Inc.) running at 2 ml/mm. The column was washed with 15 mlof Buffer A and eluted with a linear gradient ranging from 0% B (i.e.,Buffer B is an aqueous 3 M NaCl solution) to 40% B, over a time frame of12 minutes. The column was then washed with 100% B for 5 minutes.Subsequently, the column was re-equilibrated with Buffer A. Absorbancewas monitored at 280 nm to detect elution of aromatic amino acids (i.e.,Tyrosine) found in bacterial proteins.

The results demonstrate a mixture of proteins, the majority of whichelute at 0.1M to 0.8 M NaCl, and a minor fraction of material whichelutes at a 3.0 M NaCl concentration. Fractions were collected andsaved, and dialyzed in Spectrapor dialysis membranes (MW “cut-off”approximately 1,000 Daltons) against water, to facilitate subsequentanalysis.

Ultraviolet and Visible Spectroscopy:

Differential absorbance spectra were determined between 200 and 600 nmwavelengths in 1 cm quartz cuvettes using a Uvikon 930 scanningspectrophotometer (Kontron Instruments). The baseline was determinedwith water or culture media.

The results with a water blank showed an absorbance peak at 290 nm to305 nm for Bacillus coagulans, with a significant amount of additionalabsorbing material found between 210 nm and 400 nm. There was alsodemonstrated to be significant absorbance in the UV wavelengths,primarily due to presence of protein.

SDS Polyacrylamide Gel Electrophoresis:

Electrophoresis was performed by the method of Laemmli (see, Laemmli,1970. Nature 227: 680-685) and the acrylamide gels were poured in 1 mmcassettes (Novex) and run according to recommendations of the commercialsupplier (i.e., 120 volts, for 90 minutes [12% gel] and for 2 hours[16%]). The gels were then silver stained by the method of Blum, et al.(see Blum, et al., 1987. Electrophoresis 8: 93-99). A 16% acrylamide gelwas found to be best resolving the Bacillus coagulans proteins. Allsamples were dialyzed against water prior to preparation forelectrophoresis so as to ameliorate salt-associated electrophoreticartifacts. Wide range protein markers (BioRad) were used for proteinmolecular weight determination.

The electrophoretic results demonstrated a significant number ofproteinaceous bands in the ≦4,000 to 30,000 Dalton range for Bacilluscoagulans.

High Pressure Liquid Chromatography:

Five ml of culture supernatants were extracted with 2 ml ofacetonitrile, benzene, or 24:1(v:v) chloroform:isoamyl alcohol forapproximately two hours. The phases were allowed to separate for fourhours and further separated by centrifugation at 5,000×g for 10 minutes.The organic phase was then filtered through 0.2 μm PVDF filters (GehnanAcrodisc LC-13) and loaded on an Econosil C-18 10U HPLC column (Altech)in a mobile phase of 20 mM Tris-HCl (pH 7.5). Elution was started aftera total of 5 minutes, in a 15 minute linear gradient to 60% acetonitrile(ACN) in water. Elution was continued for 5 minutes in 60% ACN, and thecolumn was then washed and re-equilibrated in 20 mM Tris-HCl (pH 7.5).

The results of reverse-phase HPLC of ACN-extracted Bacillus coagulanssupernatant demonstrated that increasing the organic character of thesolvent led to increasingly “organic profiles” in the HPLC (i.e., anincrease in material eluting at higher percentage of ACN) and anincrease in the capture of pigmented molecules (i.e., molecules whichabsorb visible light). These aforementioned molecules will be isolatedand further characterized.

The results of these aforementioned analytical methodologiesdemonstrated that the culture supernatants from Bacillus coagulans isvery heterogeneous in nature, containing a plurality of proteinaceousand organic molecules. However, the molecules which predominate are theproteins, of which there are a total of 20 distinct species in each ofthe samples. These protein species can be further fractionated by use ofion exchange chromatography, thus allowing additional characterization.Furthermore, there are also numerous pigmented molecules (i.e.,molecules which absorb visible light) that are both highly conjugated(based upon their absorbance at high wavelengths) and hydrophobic (basedupon their preference for non-polar solvents and retention on the C-18HPLC column).

In an embodiment of the present invention, the liquid containing theextracellular product may be formulated into a liquid ointmentcomposition for use in direct application onto dermal, cuticular, ormucous membrane tissues. The liquid ointment was prepared by combiningthe liquid extracellular product produced above with, e.g., Emu Oil in aratio of approximately 8:2.

Isolation and Characterization of Novel Strains of Bacillus coagulans

Viable Bacterial Colony Isolation and Characterization

Dilution and Heat Treatment

Approximately 1 g of a lyophilized Bacillus coagulans sample was placedinto a surface-sterilized, homogenization container. Approximately 200ml of sterile physiological saline diluent solution, comprising 8.5 gsodium chloride and 25 mg sodium lauryl sulfate per liter, was thenadded and the mixture was homogenized at 12,000-15,000 rpm for 5minutes.

One ml of the homogenized suspension was then transferred into 9.0 ml ofsterile physiological saline in a screw-capped tube (25 mm×150 mm size)and mixed thoroughly. This serial dilution was repeated until a final2×10⁻⁸ dilution was obtained which was designated the “dilution factor.”The final diluted tube was then heat-treated in a 70° C. water-bath for30 minutes, followed by immediate cooling to 45° C.

Plating

Glucose Yeast Extract (GYE) agar medium was liquefied and then cooled to45° C. in a water-bath. A total of 5 petri dishes per sample wereutilized. 1 ml from heat-treated final dilution tube was added into eachpetri dish, followed by the addition of 5 ml of the above-identifiedliquefied GYE agar medium into the petri dishes and thorough mixing.When solidified, the plates were incubated in an inverted position at40° C. for a total of 48 hours.

Counting of Viable Bacterial Colonies

The plates showing 30-300 colonies were selected for counting. Platespossessing a very narrow variation in total colony count were countedand then an average count per plate was calculated. The number of viablecells per gram of sample was obtained by multiplying the average numberof colonies counted per plate by the reciprocal of the dilution factor(e.g., if the average number of colonies per plate was 90 and finaldilution factor was 2×10⁻⁶, then viable spore count was 90×(2×10⁶) or1.8×10¹⁰ viable spores per gram.

As will be discussed, infra, subsequent Gas Chromatography Fatty AcidMethyl Ester (GC-Frame) and Biolog™ analyses showed these bacteria to beheretofore uncharacterized strains of Bacillus coagulans. Table 8,below, illustrates the differences between the novel strains of Bacilluscoagulans disclosed herein (i.e., the 20° C. Bacillus coagulans isolate(5937-20° C.); 30° C. Bacillus coagulans isolate (5937-30° C.); the ATTC99% Bacillus coagulans isolate (ATCC-99%); and the ATTC 1% Bacilluscoagulans isolate (ATCC-1%), wherein (−) indicates no growth; (+)indicates light or minimal growth; and (++) indicates excellent oroptimal growth. Glucose Yeast Extract (GYE) media and Trypticase SoyBroth (TSB) culture media were used.

TABLE 8 Strain Media Type Incubation Temperature 30° C. 35° C. 40° C.ATCC-99% Isolate GYE − TSA − + + 1% Isolate (GBI-1) GYE TSA − − +5937-20° C. Isolate (GBI-20) GYE + TSA ++ 5937-30° C. Isolate (GBI-30)TSA ++ − + ++ ++ ++

FIG. 1 illustrates, in histogram form, the minimal and optimal culturetemperatures for the Bacillus coagulans 1% isolate (GBI-1); ATCC-99%isolate; the 5937-20° C. isolate (GBI-20); and the 5937-30° C. isolate(GBI-30), in either Trypticase Soy Broth (TSA) or Glucose Yeast Extract(GYE) media.

pH Kinetic Studies

Materials and Methods:

A total of four cultures of Bacillus coagulans strains were analyzedwith pH Kinetic Testing, Heterotrophic Plate Counts, and Optical Density(OD) in % Optical Transmittance of culture growth at 4 hour intervalsfor 28 hours in tryptic soy broth (TSB) media. These stains included:the 20° C. Bacillus coagulans isolate (GBI-20); 30° C. Bacilluscoagulans isolate (GBI-30); the ATTC 99% Bacillus coagulans isolate(ATCC-99%); and the 1% Bacillus coagulans isolate (GBI-1).

Each of the aforementioned bacterial stains were placed in 50 mlErlenmeyer flasks containing 20 ml of TSB media. Seven flasks wereprepared for each of the four isolates, one for each 4 hour interval ofthe 28 hour study. Initial seed cultures were broth cultures in testtubes, which had a % transmittance of 10%. 1.0 ml of this culture wasthen place into each of the 28 total flasks, representing 7 flasks foreach strain. These inoculated flasks were incubated on a rotaryenvironmental shaker at 45° C. for 28 hours. Every 4 hours, the shakerwas stopped, and the new culture removed for evaluations. OD readings in% Optical Transmittance, pH, and Total Heterotrophic Plate Counts by 3MPetrifilm spread plate method performed to monitor bacterial celldensity and pH changes at these different time intervals. The results ofthe pH evaluations, OD in % Transmittance, and Total Heterotrophic PlateCounts are shown below in Table 9, Table 10, and Table 11, respectively.

TABLE 9 Data: pH readings of broth culture PH GBI-20 GBI-30 ATTC-99%GBI-1  4 Hour 7.0 7.0 7.0 7.1  8 Hour 6.4 6.4 6.6 6.5 12 Hour 6.6 6.46.4 6.5 16 Hour 6.4 6.5 6.4 6.5 20 Hour 6.8 7.0 6.9 6.9 24 Hour 7.6 7.47.4 7.3 28 Hour 7.6 7.9 7.8 7.8

TABLE 10 Data: % light transmittance readings through broth cultureGBI-20 GBI-30 ATTC 99% GBI-1  4 Hour 68  51  62  65   8 Hour 8   6.5 8 812 Hour 4   3.0   3.5 4 16 Hour 40* 38* 36* 44* 20 Hour 37* 33* 33* 37*24 Hour 50* 31*   34.5* 34* 28 Hour 33* 32* 30* 34* *1:10 dilution ofbroth culture in sterile distilled water

TABLE 11 Total Heterotrophic Plate Counts by Spread Plate Method of 10⁶Dilution GBI-20 GBI-30 ATTC 99% GBI-1  4 Hour 2.2 × 10⁷ 3.9 × 10⁷ 2.5 ×10⁷ 1.7 × 10⁷  8 Hour 4.9 × 10⁷ 3.6 × 10⁸ 1.8 × 10⁸ 3.6 × 10⁸ 12 Hour7.2 × 10⁸ 2.0 × 10⁸ 9.0 × 10⁸ 9.0 × 10⁸ 16 Hour TNTC TNTC TNTC 5.2 × 10⁸20 Hour 5.0 × 10⁸ 2.4 × 10⁸ 9.9 × 10⁷ 3.4 × 10⁸ 24 Hour 3.0 × 10⁸ 3.0 ×10⁸ 1.1 × 10⁸ 3.8 × 10⁸ 28 Hour 7.0 × 10⁸ 9.2 × 10⁷ 2.8 × 10⁸ 6.8 × 10⁸

Experimental Results

As may be ascertained from Tables 9-11, there are distinct variancesbetween all isolates in regard to L(+) lactic acid production atdifferent intervals. The interval corresponding cell density wasdetermined using light transmittance using a Vitek machine operating ateither 540 or 680 nm and a standard plate count on TSB. The 20° C.Bacillus coagulans isolate (GBI-20) and the 30° C. Bacillus coagulansisolate (GBI-30) provided higher growth rates and were markedly moreefficient at lowering the pH of the fermentation broth at the 8 hourinterval and afterwards (using TSB as a fermentation substrate), thanthe 1% Bacillus coagulans isolate (GBI-1) and the ATTC 99% Bacilluscoagulans isolate. These results would seem to indicate that theseaforementioned strains are effective in mitigating diseases that arepH-specific such as Escherichia, Campylobacter, Candida, Clostridium,and Staphylococcus, than either the 1% Bacillus coagulans isolate(GBI-1) and the ATTC 99% Bacillus coagulans isolate.

Growth/End-Point Kinetic Studies

GBI-1 and ATCC-99% Bacillus coagulans Isolates

Two cultures of Bacillus coagulans strains were analyzed withGrowth/End-Point Kinetic Testing. These stains included: the 1% Bacilluscoagulans isolate (GBI-1) and the ATCC-99% Bacillus coagulans isolate.

In Kinetic Assays No. 1, the 1% Bacillus coagulans strain (GBI-1) wastested. In Kinetic Assay No. 2, the ATCC-99% Bacillus coagulans strainwas tested. The specific strain of Bacillus coagulans to be tested wasgrown for a total of 48 hours in Trypticase Soy Broth (TSB) medium at45° C. Following incubation, the cultures were suspended in sterilesaline to a turbidity (T) of approximately 40-50% T. The dilutedcultures were placed into the wells of a 96-well microtiter plate whichcontained a specific growth medium which comprised one of the following:TSB, Glucose Yeast Extract (GYE) medium, or, either with or withoutadditional oxygenation. To oxygenate the growth medium, 100 ml of eachmedium was placed under the flow of an oxygen concentrator at a rate of4 liters/minute for a total of 15 minutes. In addition, each microplatewell also contained a tetrazolium dye/redox indicator system. Bacterialgrowth (i.e., metabolic respiration or oxidation of carbon sources) wasmonitored by tetrazolium reduction as measured at 590 nm in aspectrophotometric microplate reader.

Bacterial growth was measured every 20 minutes during a total incubationof 22 hours at 32° C. The kinetic data was processed and the backgroundblank values subtracted.

Following completion of the above-referenced Kinetic Growth Assay, thetetrazolium reduction as measured at 590 nm in the microplate is read asan End-Point Kinetic assay. FIG. 2 and FIG. 3 show the End-PointKinetics of both the 1% Bacillus coagulans strain (GBI-1) and theATCC-99% Bacillus coagulans strain, respectively.

5937-20° C. and 5937-30° C. Bacillus coagulans Isolates

Two cultures of Bacillus coagulans strains were analyzed withGrowth/End-Point Kinetic Testing. These stains included: the 20° C.Bacillus coagulans isolate (GBI-20) and the 30° C. Bacillus coagulansisolate (GBI-30).

In Kinetic Assays No. 3, the 20° C. Bacillus coagulans isolate (GBI-20)was tested. In Kinetic Assay No. 4, the 30° C. Bacillus coagulansisolate (GBI-30) was tested. The specific strain of Bacillus coagulansto be tested was grown for a total of 48 hours in Glucose Yeast Extract(GYE) medium at 35° C. Following incubation, the cultures were suspendedin sterile saline to a turbidity (T) of approximately 40-50% T. Thediluted cultures were placed into the wells of a 96-well microtiterplate which contained a specific growth medium which comprised one ofthe following: GYE or Trypticase Soy Broth, Nutrient Broth (NB), orBiolog Universal Growth Medium (BUGMB), either with or withoutadditional oxygenation. To oxygenate the growth medium, 100 ml of eachmedium was placed under the flow of an oxygen concentrator at a rate of4 l/minute for a total of 15 minutes. In addition, each microplate wellalso contained a tetrazolium dye/redox indicator system. Bacterialgrowth (i.e., metabolic respiration or oxidation of carbon sources) wasmonitored by tetrazolium reduction as measured at 590 nm in aspectrophotometric microplate reader.

Bacterial growth was measured every 20 minutes during a total incubationof 18 hours at 32° C. The kinetic data was processed and the backgroundblank values subtracted.

Following completion of the above-referenced Kinetic Growth Assay, thetetrazolium reduction as measured at 590 nm in the microplate is read asan End-Point Kinetic assay. FIG. 4 and FIG. 5 represent histograms ofthe End-Point Kinetics of the 5937-20° C. Bacillus coagulans isolate(GBI-20) and 5937-30° C. Bacillus coagulans isolate (GBI-30),respectively.

Biolog™ Analysis of Bacillus coagulans Isolates

In order to differentiate the ATCC-type strain Bacillus coagulans Hammer(ATCC No. 31284) from the novel strains of Bacillus coagulans disclosedherein, the Biolog Microplate System™ was utilized for microbialidentification and characterization by carbon source patternrecognition. An innoculum of the ATCC-type strain (ATCC No. 31284) wasplaced into each of three flasks of Trypticase Soy Broth (TSB). Theseflasks were then incubated at different temperatures to compensate forany bacterial selection resulting from temperature. After 30 hrs ofincubation, an aliquot from each broth flask was aseptically transferredin a laminar flow biological cabinet and plated onto previously preparedand dried TSA medium in Petri plates. Observations for colony formingunits (CFU) are made after 24 and 48 hours of incubation at 30° C., 35°C., and 40° C.

The Biolog Microplate System™ was utilized for microbial identificationand characterization by carbon source pattern recognition of theBacillus coagulans strains disclosed in the present invention. Theaforementioned microplate technique allows for microbialcharacterization by use of 95 different analytical methods, thusyielding a total of 4×10²⁸ possible patterns generated from a singlemicroplate. Each strain of microorganism yields a distinct pattern, andthe different species of bacteria will give different “families” ofpatterns which can be recognized and differentiated by the BiologMicrolog™ software. Analytical microplates for the Biolog Microlog™system are available for gram-negative bacteria, gram-positive bacteria,yeast, lactic acid-producing bacteria, and E. coli/Salmonella analysis.In addition, further analyses may also be performed by use of additionalselective media.

In brief, characterization of a given microbial isolate is performed bystreaking the organism onto a nutrient medium (e.g., GYE or TSA) thatwill support vigorous microbial proliferation and growth. However, themore fastidious organisms may require chocolate or BIER agar for growth,whereas many “environmental” were found to grow better in the moreminimal media. The culture plates were incubated at 28° C. to 35° C. for4-18 hours.

Following incubation, the bacterial colonies were removed from theculture plate by use of a saline-moistened, cotton swab. A suspension ofuniform turbidity was then prepared in 0.85% saline by comparison with aknown turbidimetric standard. The bacterial suspension was inoculatedinto the microplate wells (150 μl/well) and the plate was covered withthe associated microplate lid. The covered plates were then incubated at28° C. to 35° C. for 4 hours or overnight (16-24 hours).

The microplates were then read using a microplate reader at 590 nm. Theabsorbence or transmittance (i.e., color) in each well was referencedagainst the negative control well (A-1) so that any purple colorrecorded above this control level was read as a positive utilization ofthat particular carbon source. The data were reported as the PercentColor Change as compared to well A-1 utilizing the following formula:

${\% \mspace{14mu} {Color}\mspace{14mu} {Change}} = \frac{{{OD}_{590}({well})}\mspace{14mu} {{OD}_{590}( {{well}\mspace{14mu} A\text{-}1} )}}{{OD}_{590}( {{well}\mspace{14mu} A\text{-}1} )}$

Generally, if the Percent Color Change was found to be equal to, orgreater than 40, the reaction within the given well was considered to be“positive”. However, this value must be empirically determined, as theparameters for each substrate may be different and the positive testbelow a value of 40 may be possible. The computer algorithms employedprovide standardization of settings ensuring repeatability and avoidanceof operator bias. Names of all carbon source substrates employed areprovided in the results regardless of response.

Table 12, below, illustrates the Total Heterotrophic Plate Count usingTrypticase Soy Agar (TSA) for the novel Bacillus coagulans strainsdisclosed herein.

TABLE 12 DATA: Direct Count, Colony Forming Units (CFU/ml) on TSA Sample24 Hours 4 Days Types GBI-1 3.00 × 10⁶ 2.00 × 10⁷ 2 GBI-20 <1.00 × 10⁶  5.74 × 10⁶ 1 GBI-1 spore shock 9.30 × 10⁸ 4.00 × 10⁹ 4 GBI-20 sporeshock 7.20 × 10⁹ 7.27 × 10⁹ 4Total morphologically different types among samples: 5

Table 13, below, illustrates the approximate percentages of aerobicstrain types in each of samples comprising the novel strains of Bacilluscoagulans disclosed herein.

TABLE 13 Sample GBI-1 spore GBI-2 spore Strain GBI-1 GBI-20 shock shock6022-1 50 5 6022-2 50 100 5 6022-3 20 15 6022-4 55 60 6022.5 20 20

GC-FAME Processing:

The bacterial strains were streaked onto Trypticase Soy Agar (TSA)plates. The TSA plates were then prepared for Gas Chromatography FattyAcid Methyl Ester (GC-FAME) analysis following a 24 hour incubation bystandard, published GC-FAME methodologies. The bacterial strain wassubsequently examined against both the Aerobe (TSBA) and the ClinicalAerobe (CLIN) computer databases. The results of the GC-FAME analysis isshown below, in Table 14.

TABLE 14 Primary ID by Dist. Strain GC-FAME Aerobic Method Sim. Coef.Coef. ATCC-99% Bacillus coagulans .533 3.927 GBI-1 Bacillus coagulans.568 3.780 GBI-20 Bacillus coagulans .542 4.176 GBI-30 Bacilluscoagulans .543 3.927 GBI-40 Bacillus coagulans .501 4.17416S Ribosomal RNA (rRNA) Sequence Analysis

Materials and Methods

Sequence analysis of 16S Ribosomal RNA (rRNA) was performed for Bacilluscoagulans strains: GBI-1; ATCC-99%; GBI-40; GBI-30; and GBI-20.

The protocol used to generate the 165 rRNA gene sequence data is setforth below. The 16S rRNA gene was PCR amplified from genomic DNAisolated from bacterial colonies. The primers which were utilized forthe amplification correspond to E. coli positions 005 and 531 for the500 by package. Excess primers and dNTPs were subsequently removed fromthe amplification products by use of a Microcon 100™ (Amicon) molecularweight cut-off membranes. The PCR amplification products were thensubjected to agarose gel electrophoretic analysis to ascertain bothquality and quantity of these products.

Cycle sequencing of the 16S rRNA amplification products was performedusing AmpliTaq FS™ DNA polymerase and dRhodamine dye terminators. Excessdye-labeled terminators were removed from the sequencing reactions usinga Sephadex G-50 spin column. The amplification products were thencollected by centrifugation, dried under vacuum, and stored at −20° C.until use. The products were resuspended in a solution of formamide/bluedextrin/EDTA, and heat-denatured prior to electrophoresis. The sampleswere electrophoresed on a ABI Prism 377 DNA Sequencer using apre-poured, 5% Long Ranger™ (RMC) polyacrylamide/urea gel forapproximately 6 hours. The resulting sequence data was analyzed usingPE/Applied Biosystems DNA editing and assembly software.

The bacterial identifications which were assigned were based upon 16SrRNA gene sequence homology. The sample sequences were identified bycomparison against PE Applied Biosystem's MicroSeg™ database utilizingMicroSeg™ sequence analysis software. Sequence alignments which providedthe highest degree of sequence homology are presented in a percentgenetic distance format (i.e., the percent difference between twoaligned sequences). It should be noted that, in this format, a lowpercentage indicates a high degree of sequence homology. FIG. 6 throughFIG. 8 provides alignment of the novel Bacillus coagulans strainsdisclosed in the present invention with various other Bacillus species,as well as the results obtained by Neighbor Joining Tree and ConciseAlignment analysis. The results for the ATCC-99% isolate are shown inFIG. 6; results for GBI-20 are shown in FIG. 7; and results for GBI-30are shown in FIG. 8.

Also provided herein are nearest neighbor (see, Saitou and Nei, 1987.Mol. Biol. Evol. 4: 406-425) and/or UPGMA (see, Waterman, 1995. In:Introduction to Computational Biology, p. 360-365 (Chapman and HallPublishing)). Similarly, the “trees” were generated using the alignmentsequences matches providing the highest degree of sequence homology.

Experimental Results

It should be noted that all experimental results are presented in agenetic distance format, which is essentially the opposite of percenthomology.

Species Level:

This indicates a species level match. A 16S rRNA sequence homology ofgreater than 99% is indicative of a species level match (see,Staekebrandt and Goebel, 1994. Taxonomic Note: A Place for DNA-DNAReassociation and 16S rRNA Sequence Analysis in the Present SpeciesDefinition in Bacteriology. Int. J. Syst. Bacteriol. 44: 846-849).

Genus Level:

This indicates that the sample appears to group within a particulargenus but the alignment did not produce a species level match. A genuslevel match indicates that the sample species is not included in theMicroSeq database.

No Match:

This indicates that sample did not group well within any particulargenus found in the MicroSeq database. In cases such as this, a search ofboth the GenBank and Ribosomal Database Project (RDP) databases with thesample sequence was subsequently performed to try to provide a closermatch. If the sample sequence does not match well with either of thesedatabases, it may represent a new species or a species whose 165 rRNAgene sequence is not present in any of the databases.

Table 15, below, provides the results of the Percent Genetic Differencestudies in tabular form.

TABLE 15 Strain No. Identification % Difference Confidence Level GBI-1Bacillus coagulans 1.68% difference Genus level ID ATCC-99% Bacilluscoagulans 1.68% difference Genus level ID GBI-40 Bacillus coagulans1.68% difference Genus level ID GBI-30 Bacillus coagulans 1.68%difference Genus level ID GBI-20 Bacillus coagulans 1.68% differenceGenus level ID

The 16S rRNA sequence homology was found to be greater than 99% andindiciative of a species level match.

Aminopeptidase Profiling

Aminopeptidase profiling or activity has been used to differentiatebacteria and fungi to species and sub-species (see, e.g., Hughes, etal., 1988. LacZY gene modified peptidase activity in Pseudomonasaureofaciens. Phytopathology 78: 1502; Hughes, et al., 1989.Identification of immobilized bacteria by aminopeptidase profiling.Anal. Chem., 61: 1656-1660), as well as to define ecological niches ofparasites and develop media for fastidious organisms. The recentdevelopment of a time-resolved, 96-well plate fluorometer provides arapid and highly sensitive method to obtain peptidase profiles formicrobial identification. See, Mossman, et al., 1997. Aminopepetidaseprofiling using a time-resolved, 96-well plate filter fluorometer. Appl.Spectroscopy 51: 1443-1446.

Aminopeptidase profiling was shown to be an effective procedure for thedifferentiation of the novel strains of Bacillus coagulans disclosedherein, from those previously known and characterized (e.g., the ATCCtype strain).

Materials and Methods

The Aminopeptidase profiling analysis disclosed follows themethodologies as set forth by Mossman, et al., 1997. Appl. Spectroscopy51: 1443-1446. Each Bacillus coagulans isolate was initially cultured onTryptic Soy Broth (TSB) Agar plates for 24 hours before washing from theplate with 10 mM, pH 7, phosphate buffer. Table 16, below, illustratesthe culture conditions of the various strains of Bacillus coagulanswhich were utilized in the present invention.

TABLE 16 Growth Temp Ref No. Strain Name for Profiling Comments Plate 1ATCC-99% 45° C. 99% majority isolate from ATCC #31284 culture. Plate 2GBI-1 45° C. Plate 3 GBI-30 45° C. Strain grew well at 30° C. Plate 4GBI-20 45° C. Strain grown on the bench top at 20° C.

Following culture, the cell densities were adjusted to 2.5×10⁶ cells/mlby spectrophotometry at 540 nm (85% transmittance) before placing 0.5 mlinto each cell of a 96-well, flat bottom, black, polystyrene plate(FluoroNunc; Nalge-Nunc, Naperville, Ill.). Each well contained one of20 different non-fluorescent, L-amino acid-β-naphthylamide substrates(Sigma Chemical Co., St. Louis, Mich.) at a final concentration of1×10⁻⁴ M. The balance of the microplate well volume of 300 μl consistedof 250 μl of the 10 mM phosphate buffer.

The 20 different peptidase substrates used to produce the profilesincluded β-naphthylamides of the following amino acids: L-alanine (ALA),L-arginine (ARG), L-asparagine (ASN), L-aspartic acid (ASP), L-cysteine(CYS), glycine (GLY), L-glutamic acid (GLU), L-histidine (HIS),L-isoleucine (ILE), L-leucine (LEU), L-lysine (LYS), DL-methionine(MET), L-phenylalanine (PHE), L-proline (PRO), L-serine (SER), transhydroxy-L-proline (HPR), L-tryptophan (TRP), L-tyrosine (TYR), andL-valine (VAL). β-naphthylamine, alone, was also used as a positivecontrol. A bacterium blank, substrate blank, and buffer blank were alsoincluded in the assay procedure as negative controls. Four replicationsof each bacterial isolate were run after a 4-hour incubation period.

Aminopeptidase profiles were constructed with data obtained from atime-resolved, laser fluorometric assay of the enzymatically hydrolyzed,fluorescent, β-naphthylamide product from the non-fluorescent,β-naphthylamide substrates. The time-resolved, 96-well plate fluorometerconsisted of a sealed tube, nitrogen laser that is guided to a black,flat bottom FluoroNunc 96-well plate via the excitation portion of abifurcated fiber optic. Fluorescence was collected at a 0° angle to theexcitation beam with the emission portion of the bifurcated fiber optic.A 389 nm cut-on filter was used to select the desired emissionwavelength before detection with a 931 A photomultiplier tube. A totalof 25 fluorescent decays were averaged by a Tektronix DSA 602 digitaloscilloscope and transferred to a PC computer via an IEEE-488 interfacecard to provide a readout of relative fluorescence after blanksubtraction.

Experimental Results:

Significant differences are detected in the enzyme profile of theseBacillus coagulans strains which otherwise are identical for 16S rRNASequencing, GC-FAME, and Biolog Identifications. The data is presentedfor each of the four Bacillus coagulans strains in a histogram formatplotting fluorescence intensity for each the aminopeptidase enzymeactivities listed below. FIG. 9 represents a histogram plot of the ofthe fluorescence intensity for each the aminopeptidase enzyme activitiesfor the Bacillus coagulans 99% ATCC isolate; FIG. 10 represents ahistogram plot of the of the fluorescence intensity for each theaminopeptidase enzyme activities for the Bacillus coagulans GBI-1isolate; FIG. 11 represents a histogram plot of the of the fluorescenceintensity for each the aminopeptidase enzyme activities for the Bacilluscoagulans GBI-30 isolate; and FIG. 12 represents a histogram plot of theof the fluorescence intensity for each the aminopeptidase enzymeactivities for the Bacillus coagulans GBI-20 isolate. Each of thespecific Aminopeptidases and controls, as set forth in FIG. 9 throughFIG. 12, are identified using numbers 1-24. These numbers are asfollows:

1. L-alanine (ALA) 13. L-methionine (MET) 2. L-asparagine (ASN) 14.L-phenylalanine (PHE) 3. L-arginine (ARG) 15. L-proline (PRO) 4.L-aspartic acid (ASP) 16. trans-hydroxy-L-proline (HPR) 5. L-cysteine(CYS) 17. L-serine (SER) 6. L-glutamine (GLN) 18. L-threonine (THR) 7.L-glutamic acid (GLU) 19. L-tryptophan (TRP) 8. L-glycine (GLY) 20.L-tyrosine (TYR) 9. L-histidine (HIS) 21. L-valine (VAL) 10.L-isoleucine (ILE) 22. β-napthylamine (Positive Control) 11. L-leucine(LEU) 23. Buffer (Negative Control) 12. L-lysine (LYS) 24. Buffer withCells (Negative Control)Activity for Numbers 12 (Lysine aminopeptidase) and 22 (β-Napthylamine,100% control) are not plotted when found to be “off-scale”. All celldensities were standardized at 85% T.

The results, illustrated in FIG. 9 through FIG. 12, demonstrate thatdifferences exist in the Aminopeptidase profiles of these Bacilluscoagulans isolates, despite the overall similarity within the profiles.For example, the 20° C. isolate GBI-20 (see, FIG. 12) is most similar tothe 99% isolate (see, FIG. 9) with a dramatic departure in the relativeamount of Proline aminopeptidase; whereas the 30° C. isolate GBI-30(see, FIG. 11) more closely resembles the pattern of the 1% isolateGBI-1 (see, FIG. 10), but departs in the relative amount ofPhenylalanine aminopeptidase. Thus, it appears that this methodology maybe utilized to both rapidly and effectively differentiate these Bacilluscoagulans strains.

Use of Bacillus coagulans in the Inhibition of Gastrointestinal VRE

The ability of Bacillus coagulans vegetative bacteria and spores toinhibit the colonization of Vancomycin-Resistant Enterococci (VRE) wasexamined. Prior to the disclosure of the present invention, no effectivetherapy was available to decrease either the amount or the duration ofintestinal colonization with VRE. For example, many antibiotics havebeen shown to have only a very transient effect on VRE colonization.Thus, the development of a safe and efficacious therapeutic for theamelioration of VRE colonization would serve to significantly reduce thepotentially fatal consequences of VRE infection, the transmission of VREbetween patients hospital costs, and patient and healthcare-providerinconvenience.

Materials and Methods

A murine model, initially developed to study the effect of variousantibiotics on persistence of VRE intestinal colonization, was used inthese experiments. Two sets of experiments, using a total of 33 micewere performed. High-level VRE colonization was established in all 33mice by administering approximately 5×10⁸ VRE by oral gavage, whileconcurrently administering subcutaneous Clindamycin daily for 5 days.This method consistently results in development of high levels of VREfecal colonization in mice (mean=9 log₁₀ CFU/gram of stool).

The mice were then divided into 3 experimental groups and the followingagents were administered: Group 1=saline by oral gavage for 4 days (11total control mice); Group 2=Bacillus coagulans overnight cultureapproximately 1×10⁷ vegetative organisms by oral gavage for 4 days (17total mice); and Group 3=Bacillus coagulans spores approximately 1×10⁷organisms by oral gavage for 4 days (5 total mice). Stool samples werecollected at 3 to 5 day intervals during the experiment to determine thelevels of VRE and Bacillus coagulans. Stool samples were homogenized,serially diluted in saline, and plated on enterococcosel-selective agarfor quantification of VRE, or on BHI agar containing 6 μg/ml ofaztreonam and 6 μg/ml of Nystatin for quantification of Bacilluscoagulans. If VRE were not detectable in a sample, the lower limit ofdetection was assigned.

Preliminary Microbiology Results Kirby-Bauer Antibiotic SusceptibilityTesting:

-   Susceptible to: ampicillin, ciprofloxacin,    trimethoprim-sulfamethoxazole, rifampin, erythromycin, vancomycin,    gentamicin, and oxacillin-   Intermediate Susceptibility to: tetracycline

Vitek Machine-Based Susceptibility Testing:

-   Susceptible to: penicillin, vancomycin, gentamicin (500 μg/ml),    streptomycin (2,000 μg/ml), nitrofurantoin, norfloxacin, and    chloramphenicol-   Resistant to: tetracycline

Nitrocefin Testing:

-   Positive low-level β-lactamase production

Murine Conolization

Bacillus coagulans was given to eight mice to determine the doses to beused in the subsequent, formal experiments. All mice were colonized withhigh levels of VRE (>9 log₁₀CFU/gram of stool) prior to administrationof Bacillus coagulans. Control mice received no treatment. Bacilluscoagulans was administered daily by gastric gavage in three differentdoses: 1.5×10⁶ CFU/kg=usual human dose, 2.5×10⁸ CFU/kg and 3.5×10⁹CFU/kg. The level of VRE in stool was determined after 5 days. Theresults of these preliminary studies are shown below in Table 17.

TABLE 17 Mean Level of Treatment Mice Group No. VRE on Day 5 Controlmice (no treatment) 4  6.6 log₁₀CFU/g stool 5 × 10⁶ CFU/kg/day 4  6.0log₁₀CFU/g stool 5 × 10⁸ CFU/kg/day 3 *3.5 log₁₀CFU/g stool 5 × 10⁹CFU/kg/day 1  3.7 log₁₀CFU/g stool *The level of VRE was below the levelof detection (<=1.7 log₁₀CFU/g) for ⅔ mice treated with 5 × 10⁸ CFUBacillus coagulans/kg/day. The lower limit of detection was assigned tothese mice.

By use of the aforementioned colonization methodology, high-levels ofVRE colonization was initially established in all of the mice (i.e., 7.1to 10.2 log₁₀VRE/gram of stool). The initial level of VRE present in thesaline control mice and the Bacillus coagulans mice was notsignificantly different. The level of VRE declined gradually in all ofthe saline control mice after Clindamycin was discontinued (consistentwith previous experiments).

In comparison to the saline controls, the level of VRE declined morerapidly in the mice receiving Bacillus coagulans. Five days afterclindamcin was discontinued (after 4 days of Bacillus coagulanstherapy), the mean level of VRE was found to be 5.3 log₁₀VRE/gram ofstool compared with 6.7 log₁₀VRE/gram of stool in the saline controls.This represented a 25-fold reduction in VRE levels (p<0.05). Eight daysafter clindamycin was discontinued (4 days after Bacillus coagulanstherapy was completed), the mean level of VRE was found to be 2.9log₁₀VRE/gram of stool compared with 4.3 log₁₀VRE/gram of stool in thesaline controls. This represented a 28-fold reduction (p<0.05).Thirty-five percent ( 6/17 animals) of Bacillus coagulans treated micehad undetectable levels of VRE eight days after clindamycin wasdiscontinued, whereas none of the saline controls had undetectablelevels of VRE at that time point (p<0.05). The mean level of VRE presentin the stool of the 5 mice receiving Bacillus coagulans spores was alsosignificantly lower than the level in the saline control mice (p<0.05),however none of these five mice had undetectable VRE levels.

All of the mice receiving Bacillus coagulans had detectable levels ofBacillus coagulans in their stool one day after completion of four daysof therapy (range 3.1 to 6.4 log₁₀CFU/gram of stool) and all of thesemice still had low levels of Bacillus coagulans detectable in theirstool 4 days after completion of therapy.

These studies demonstrated that the oral administration of Bacilluscoagulans (in the form of both vegetative bacteria and spores) resultedin a significant decrease in the level of VRE in the stool of colonizedmice, in comparison with saline controls. The results which wereobtained with the use of this murine model correlate well with thefindings in various studies which were examined VRE-colonized humanpatients. Therefore, this established mouse model provides a means tostudy the efficacy of agents designed to eliminate VRE colonization.Thirty-five percent of mice receiving Bacillus coagulans were found tohave undetectable levels of VRE four days after completing therapy. Incomparison, none of the mice receiving saline were VRE-free. On average,a 25- to 28-fold reduction in the level of VRE was observed in theBacillus coagulans-treated mice in comparison with the saline-treatedmice. Moreover, sixty-five percent of mice receiving Bacillus coagulanshad a reduction of VRE equal to approximately 50-times the originalinoculation. Therefore, all of the test mice had a significant VRE loadreduction, with 60% of the mice exhibiting a 2-log VRE diminution and40% with complete eradication of Enterococci with statistical zeropercent recovery of VRE in the mouse stool. These results suggest thatBacillus coagulans therapy is an effective means to ameliorate both thelevel and duration of VRE colonization in human patients.

The inhibition of VRE by Bacillus coagulans does not appear to involveany of the mechanisms of inhibition traditionally though to be used byprobiotic bacteria such as Bacillus coagulans. As previously discussed,there are two primary mechanisms used by acid-producing Bacillus forelimination of microbes. These mechanisms are:

Competitive Inhibition or Exclusion:

Which is the ability of most Bacillicea to out-compete other organismsfor substrate and trace minerals. This usually involves the massproliferation of the Bacillus.

Micro-Environment Modification:

Which usually serves to alter the physiological or biochemicalproperties or activities of bacteria's cell membrane by the productionof acid (e.g., lactic, acetic, etc.) or other agents possessinganti-microbial properties.

Although there was a dramatic decrease in the VRE levels (i.e., 2-logsin the 60% effective group and 40% in the total eradication group), theresults show that there was no corresponding increase in Bacilluscoagulans concentrations of the treated groups (expressed in CFU pergram of mouse stool). It appears that one experimental group showedsubstantially better results than another successful group, but withouta corresponding Bacillus enumeration to justify it. Accordingly, theseresults suggest that Competitive Inhibition by the Bacillus coagulans isnot the mechanism which gave rise to the mitigation of VRE levels inthis study.

Additionally, it is also known that Enterococci are not inhibited bychanges in the pH of its micro-environment. For example, Enterococcusfaecium (which is the Enterococcus species responsible for most, if notall, VRE carriage and infections) is used as a probiotic in the animalproduction industry. This organism, itself, produces a D-optical isomerof lactic acid and is generally co-administered with Lactobacillus andBifidiobacterium, which produce the L-optical isomer of lactic acid.Therefore, Enterococcus faecium is not affected by lactic acid-producingorganisms, regardless of optical isomer of lactic acid produced.Accordingly, the second method used by probiotic bacteria(micro-environmental changes) to inhibit microbial colonization, doesnot appear to play a role in the inhibition of VRE by Bacilluscoagulans. Due to the aforementioned experimental results, it isbelieved that the amelioration of VRE by Bacillus coagulans is due tothe production of one or more anti-microbial agents by the Bacillus.This anti-microbial agent may be an organic molecule(s) and/or anthermo-tolerant protein(s).

A composition for inhibiting VRE growth contains a large concentration(i.e., 1×10⁹ to 1×10¹¹ CFU) of Bacillus coagulans vegetative bacteriaand/or spores in combination with the culture medium (supernatant) ineither an unpurified or semi-purified form. As with Bacillus coagulansvegetative cells and spores, the culture medium has also been designateda GRAS classification by the FDA. In order to reduce the overall volume,the medium may be partially- or fully lyophilized. Thus, the concomitantadministration of both the vegetative bacteria/spores and a supernatantcomponent of some type would serve to ensure that all possible probioticinhibitory mechanisms (i.e., antibiosis, parasitism, competitiveinhibition and microenvironment/pH modification) were covered by theadministration of the aforementioned therapeutic composition.

As previously discussed supra, Bacillus coagulans culture medium hasbeen shown to contain extracellular product(s), produced and secreted bythe bacteria, which possess marked anti-microbial properties againstbacteria, fungus, yeast, and virus. Methodologies for the purificationof the one or more agents responsible for these anti-microbialproperties are also currently under development. A preferred embodimentof the present invention would, accordingly, comprise a largeconcentration (i.e., 1×10⁹ to 1×10¹¹ CFU) of Bacillus coagulansvegetative bacteria and/or spores in combination with the either apurified or semi-purified form of these extracellular product(s).

Bacillus coagulans therapy is also useful to inhibit other strains ofVRE. Similarly, the Bacillus coagulans is used to prevent or amelioratethe level of colonization of other pathogenic organisms such as Candidaspecies, Salmonella, coagulase-negative Staphylococci, andmulti-resistant gram-negative rods such as Klebsiella species andEscherichia coli.

EQUIVALENTS

From the foregoing detailed description of the specific embodiments ofthe present invention, it should be readily apparent that a uniquemethodology for the utilization of lactic acid-producing bacteria,preferably Bacillus coagulans, for the prevention and treatment ofgastrointestinal tract pathogens and their associated diseases, has beendescribed. Although particular embodiments have been disclosed herein indetail, this has been done by way of example for purposes ofillustration only, and is not intended to be limiting with respect tothe scope of the appended claims which follow. In particular, it iscontemplated by the inventor that various substitutions, alterations,and modifications may be made to the invention without departing fromthe spirit and scope of the invention as defined by the claims. Forinstance, the choice of the particular antibiotic which is utilized inthe Therapeutic Composition of the present invention is believed to be amatter of routine for a person of ordinary skill in the art withknowledge of the embodiments described herein.

Other embodiments are within the following claims.

We claim:
 1. A composition comprising an extracellular product derivedfrom an isolated Bacillus coagulans GBI-30 strain (ATCC DesignationNumber PTA-6086).
 2. The composition of claim 1, further comprising apharmaceutically-acceptable carrier suitable for oral administration. 3.The composition of claim 1, wherein said extracellular product is asupernatant or filtrate of said isolated Bacillus coagulans GBI-30strain (ATCC Designation Number PTA-6086).
 4. The composition of claim1, wherein said extracellular product is semi-purified, purified, orlyophilized.
 5. The composition of claim 1, wherein said extracellularproduct possesses anti-microbial activity.
 6. The composition of claim2, wherein said extracellular product is present at a ratio of 1% to90%.
 7. The composition of claim 6, wherein said extracellular productis present at a ratio of 10% to 75%.
 8. The composition of claim 1,wherein said composition is formulated into a liquid ointmentcomposition.
 9. The composition of claim 8, further comprising Emu oil.10. The composition of claim 1, wherein said strain produces L(+)dextrorotatory lactic acid and produces spores resistant to temperaturesof up to 90° C.